Complex surface-bound transposome complexes

ABSTRACT

The present disclosure relates to methods, compositions, and kits for generating a library of tagged nucleic acid fragments without using PCR amplification, including methods and compositions for fragmenting and tagging nucleic acids (e.g., DNA) using transposome complexes immobilized on solid support.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

The present application claims the benefit of priority to U.S.Provisional Patent Application No. 62/791,509, filed Jan. 11, 2019, andU.S. Provisional Patent Application 62/840,610, filed Apr. 30, 2019,which are hereby incorporated by reference in their entireties.

FIELD

The present disclosure relates to methods, compositions, and kits forgenerating a library of tagged nucleic acid fragments without using PCRamplification, including methods and compositions for fragmenting andtagging nucleic acids (e.g., DNA) using transposome complexesimmobilized on solid supports.

BACKGROUND

Current protocols for next-generation sequencing (NGS) of nucleic acidsamples routinely employ a sample preparation process that converts DNAor RNA into a library of fragmented templates that can be sequenced.Sample preparation methods often require multiple steps and materialtransfers and expensive instruments to effect fragmentation, which canmake these methods difficult, tedious, expensive and inefficient.Furthermore, amplification using primers introduces bias in the librarycontent and thus into the resulting sequencing data. For example, PCRamplification steps may generate gaps, which are exacerbated in GC richregions due to the inability of polymerases to efficiently copy GC richregions. These gaps create bias in the resulting sequencing data fromthe libraries. Library preparation processes with PCR amplificationsteps may have significantly reduced insertion and deletion (indel)calling performance. Some regions with multiple expanded repeats may bedifficult to sequence accurately and DNA having GC-rich promoters mayexhibit low coverage within the genome. Furthermore, many librarypreparation methods are not compatible with incorporation of sampleindices or require multiple additional steps to introduce such indices.

Library preparation procedures that are short, efficient, and accurateare needed. Herein are described various methods, compositions, and kitsthat address these problems and that accommodate single and dualindexing approaches.

SUMMARY

The present disclosure relates to methods, compositions, and kits forgenerating a library of tagged nucleic acid fragments without using PCRamplification to incorporate the fragment tags, such as primer sequencesand/or index sequences, including methods and compositions forfragmenting and tagging nucleic acids (e.g., DNA) using transposomecomplexes immobilized on solid supports.

Some embodiments provided herein relate to a transposome complex thatincludes an attachment polynucleotide. In some aspects, the disclosurerelates to a transposome complex comprising: (a) a transposase; (b) afirst transposon including a 3′ transposon end sequence and a 5′ adaptorsequence; (c) a second transposon including a 5′ transposon end sequenceand a 3′ adaptor sequence, wherein the 5′ transposon end sequence iscomplementary to at least a portion of the 3′ transposon end sequence;and (d) an attachment polynucleotide including: (i) an attachmentadaptor sequence hybridized to one of the two adaptor sequences and (ii)a binding element. Typically, the transposon end sequences are annealedtogether, forming a double-stranded transposon end sequence that isrecognized by a transposase. In some aspects, the binding element isimmobilized to a solid support to provide an immobilized transposomecomplex.

In some aspects, the disclosure relates to an annealedtransposon/attachment polynucleotide hybrid comprising the firsttransposon, the second transposon, and the attachment polynucleotide.

In some aspects, the disclosure relates to methods of making an annealedtransposon/attachment polynucleotide hybrid comprising transposomecomplex comprising annealing the first transposon, the secondtransposon, and the attachment polynucleotide. In some aspects, thedisclosure relates to methods of making a transposome complex comprisingtreating an annealed transposon/attachment polynucleotide hybrid with atransposase. In some aspects, the method provides a method of making animmobilized transposome complex by immobilizing the transposome complexto the solid support through the binding element.

In some aspects, the disclosure relates to a transposome complexcomprising: (a) a transposase; (b) a first transposon comprising a 3′transposon end sequence and a 5′ adaptor sequence; (c) a secondtransposon comprising a 5′ transposon end sequence complementary to atleast a portion of the 3′ transposon end sequence and a 3′ adaptorsequence; and (d) a binding element attached to the 5′ adaptor sequencethrough a cleavable linker. In some aspects, the binding element isimmobilized to a solid support, providing an immobilized transposomecomplex. In some aspects, the disclosure relates to an annealedoligonucleotide construct comprising the first transposon, the secondtransposon, and the binding element.

In some aspects, the disclosure relates to methods of generating alibrary of tagged nucleic acid fragments comprising contacting a targetnucleic acid with a plurality of immobilized transposome complexes asdescribed herein under conditions sufficient to fragment the targetnucleic acid into a plurality of target fragments and to join the 3′ends of the 3′ transposon end sequences to the 5′ ends of the targetfragments to produce a plurality of 5′ tagged target fragments.

Some embodiments provided herein relate to a kit for generating alibrary of tagged nucleic acid fragments without using PCRamplification. In some embodiments, the kit includes an immobilizedtransposome complex as described herein.

In some aspects, the disclosure relates to methods of generating alibrary of tagged nucleic acid fragments comprising contacting animmobilized transposome complex with a target nucleic acid underconditions sufficient to fragment the target nucleic acid into aplurality of target fragments, and to join the 3′ end of the firsttransposon to the 5′ ends of the target fragments to produce a pluralityof 5′ tagged target fragments; treating the solid support to removeunbound nucleic acids; or treating the solid support to remove thetransposase from the complex, optionally by (a) heating the solidsupport and/or (b) washing the solid support with an enzyme denaturingagent, wherein the enzyme denaturing agent optionally comprises sodiumdodecyl sulfate (SDS), guanidine hydrochloride, urea, or proteinase;treating the plurality of 5′ tagged target fragments with a polymeraseand a ligase to extend and ligate the 5′ tagged target fragments toproduce fully double-stranded tagged fragments, optionally wherein thetreating with a polymerase and a ligase is done in the presence of a DNAsecondary structure disruptor, wherein the disruptor is optionally DMSO;removing the fully double-stranded tagged fragments from the solidsupport, optionally wherein the removing comprises applying heat and/ora denaturant sufficient to cleave the fully double-stranded taggedfragments from the solid support, optionally wherein the denaturant isNaOH; and selecting the fully double-stranded tagged fragments usingcapture beads, optionally wherein the capture beads are magnetic beads,further optionally wherein two separate selecting steps are performed.

In some embodiments, the immobilized transposome complex comprises asolid support; and a transposome complex immobilized to the solidsupport, wherein the transposome complex comprises a transposase; afirst transposon comprising a 3′ transposon end sequence and an anchorsequence (Anchor); a second transposon comprising a 5′ transposon endsequence and a B15′ sequence; and an attachment polynucleotidecomprising an anchor sequence complement (Anchor′), an A14′ sequence, aspacer, and a P5′ sequence and a binding element comprising biotin,wherein the biotin is immobilized to the solid support. In someembodiments, a method further comprises sequencing one or more of thefully double-stranded tagged fragments.

In some embodiments, a method comprises contacting a first immobilizedtransposome complex and a second immobilized transposome complex with atarget nucleic acid under conditions sufficient to fragment the targetnucleic acid into a plurality of target fragments, and to join the 3′end of each first transposon to the 5′ ends of the target fragments toproduce a plurality of first 5′ tagged target fragments generated fromthe first immobilized transposome complex and a plurality of second 5′tagged target fragments generated from the second immobilizedtransposome complex. In some embodiments, the first immobilizedtransposome complex comprises a solid support and a first transposomecomplex immobilized to the solid support, wherein the first transposomecomplex comprises a transposase; a first transposon comprising a 3′transposon end sequence and an anchor sequence; a second transposoncomprising a 5′ transposon end sequence; and a first attachmentpolynucleotide comprising (i) an anchor sequence complement, an A14′sequence, a spacer, and a P5′ sequence, and (ii) a binding elementcomprising biotin, wherein the biotin is immobilized to the solidsupport. In some embodiments, the second immobilized transposome complexcomprises a solid support and a second transposome complex immobilizedto the solid support, wherein the second transposome complex comprises atransposase; a first transposon comprising a 3′ transposon end sequenceand an anchor sequence; a second transposon comprising a 5′ transposonend sequence; and a second attachment polynucleotide comprising (i) ananchor sequence complement, a B15′ sequence, a spacer, and a P7′sequence, and (ii) a binding element comprising biotin, wherein thebiotin is immobilized to the solid support. In some embodiments, themethod comprises treating the plurality of 5′ tagged target fragmentswith a ligase to ligate each 5′ tagged target fragment to either a firstindexing oligonucleotide or second indexing oligonucleotide bycontacting the 5′ tagged target fragments with a pool of first andsecond indexing oligonucleotides, wherein each first indexingoligonucleotide comprises an A14 sequence, i5 sequence, and P5 sequenceand can associate with a first 5′ tagged target fragment; and whereineach second indexing oligonucleotide comprises a B15 sequence, i7sequence, and P7 sequence and can associate with a second 5′ taggedtarget fragment, to produce a plurality of 5′ tagged target fragmentsligated to indexing oligonucleotides; treating the solid support toremove the transposases from the complex, optionally by (a) heating thesolid support and/or (b) washing the solid support with an enzymedenaturing agent, wherein the enzyme denaturing agent optionallycomprises sodium dodecyl sulfate (SDS), guanidine hydrochloride, urea,or proteinase; and treating the plurality of 5′ tagged target fragmentsligated to indexing oligonucleotides with a polymerase to extend andproduce fully double-stranded tagged fragments. In some embodiments, thecontacting a first immobilized transposome complex and a secondimmobilized transposome complex and the treating the plurality of 5′tagged target fragments with a ligase are performed in a singlereaction. In some embodiments, the double-stranded tagged fragments areproduced in solution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram that depicts an embodiment of a method ofgenerating a library of tagged nucleic acid fragments without using PCRamplification. In a first step, a transposome complex as describedherein and immobilized on a solid support is provided. A target nucleicacid is applied to the solid support, and a tagmentation reaction takesplace, generating tagged and fragmented nucleic acids. An index mixhaving index sequences is applied, and extension and ligation takesplace. Finally, indexing of the tagged nucleic acid fragments takesplace. In some embodiments, indexing occurs simultaneously withextension and ligation and in other embodiments, indexing occurs afterextension and ligation. In other embodiments, the arrangements of stepsdiffer, such as depicted in Example 9.

FIGS. 2A-2B illustrate a schematic diagram and method of preparing alibrary of tagged nucleic acid fragments without using PCRamplification. FIG. 2A is an exemplary configuration of a transposomecomplex with a biotin (B) attached to one transposon through a cleavablelinker. In this exemplary embodiment of a transposome complex comprisinga transposase, a first transposon comprising a 3′ transposon endsequence and a 5′ adaptor sequence; a second transposon comprising a 5′transposon end sequence complementary to at least a portion of the 3′transposon end sequence and a 3′ adaptor sequence; and a binding elementattached to the 5′ adaptor sequence through a cleavable linker (FIG.2A). FIG. 2B schematically illustrates exemplary steps of a method ofpreparing a library of tagged nucleic acid fragments without using PCRamplification, using the exemplary transposome complex from FIG. 2Aimmobilized to a solid support (solid support is not shown), includingsteps of tagmenting and washing, extending and ligating, and removingbeads. FIG. 2B shows the steps of tagmentation where inserts from thetarget nucleic acid are appended with the tags, extension and ligation,and cleavage from the solid support.

FIGS. 3A-3C illustrate exemplary steps of a method of generating alibrary of tagged nucleic acid fragments without using PCRamplification. FIG. 3A depicts a transposome complex immobilized to asolid support through an attachment polynucleotide bearing a biotin (B,solid support not shown). For simplicity, the depiction of the dimer isshown in FIG. 3A, but not in FIGS. 3B-3C. FIG. 3B depicts a tagged andfragmented nucleic acid, still complexed to the transposase (top panel)and structure after removal of transposase (bottom panel). FIG. 3Bdepicts the transposome complex having a nucleic acid fragment (insert)bound to the first transposon, with the 5′ end of the insert attached tothe 3′ transposon end sequence. Transposase is removed using methodsdescribed herein, for example, with the use of an agent to removetransposase, such as with sodium dodecyl sulfate (SDS). FIG. 3C depictsthe structure after gap filling and extension (extension and ligation)(top panel), and after dehybridization to remove the generated fragmentsfrom the solid support (bottom panel). As shown in FIG. 3C, indices areadded to the solid support, and hybridize to the attachmentpolynucleotide of the second transposon. Specifically, as shown in theembodiment of FIG. 3C, an i5 index having a primer sequence (P5sequence), an index sequence (i5 sequence), and an anchor sequencehybridizes to the attachment polynucleotide at complementary sequences,such that P5 hybridizes to P5′, and anchor hybridizes to anchor′.Similarly, in the embodiment of FIG. 3C, an i7 index having a primersequence (P7), an index sequence (i7), and an adaptor sequence (B15sequence) hybridizes to the second transposon at complementarysequences, such that the P7 hybridizes to P7′, the i7 hybridizes to i7′,and B15 hybridizes to B15′. After contacting the solid support with theindices, the fragments are extended and ligated using an extension andligation mix (ELM). The solid support is then treated with an agent todenature the strand sequences, such as with NaOH, thereby generatingtagged nucleic acid fragments. An embodiment of a transposome complex isshown, having a Tn5 transposase with a first and second transposon. Thefirst transposon includes a 3′ transposon end sequence (ME sequence)hybridized to a 5′ transposon end sequence (ME′ sequence) of the secondtransposon. The second transposon also includes a 3′ adaptor sequence(B15′ sequence). The first transposon includes a 5′ adaptor sequence(A14 sequence), which is shown hybridized to an attachment adaptorsequence (A14′ sequence) of an attachment polynucleotide. The attachmentpolynucleotide also includes an anchor sequence (anchor′), a spacerregion, a primer sequence (P5′ sequence), and a linker attached to abinding element (B). The transposome complex is a dimer, having twotransposome monomers dimerized.

FIGS. 4A-4E illustrate a schematic diagram comparing variousconfigurations of the transposome complex immobilized to a solid supportvia an exemplary biotin (B, solid support not shown), including indexedbeads and universal beads, each shown in both single indexing and dualindexing modes, for embodiments of methods of generating a library oftagged nucleic acid fragments without using PCR amplification, usingvariations of transposome complexes, wherein components of thetransposons and/or attachment polynucleotides are altered, arranged, orvaried in comparison to one another. FIG. 4A depicts the variousconfigurations of the transposome complex attached to a solid support.FIG. 4A depicts embodiments of the transposome complexes showingtransposase with a first and second transposon, and attached to a solidsupport through the attachment polynucleotide. FIG. 4B depicts thevarious configurations after tagmentation. FIG. 4B depicts thetagmentation reaction for indexed or universal beads with single or dualindexing, where the solid support is contacted with nucleic acidfragments (insert), which bind to the 3′ transposon end of the firsttransposon. FIG. 4C depicts the various configurations after extensionand ligation. FIG. 4C depicts extension and ligation for indexed oruniversal beads with single or dual indexing, where the solid support iscontacted with an index mix, which hybridize to the transposon orattachment polynucleotide, and wherein the nucleic acid fragments areextended. FIG. 4D depicts the various configuration after indexing (theindexed beads for single indexing is not shown, as indexing wascompleted for this configuration in FIG. 4C). FIG. 4D depicts indexingfor indexed beads with dual indexing or universal beads with single ordual indexing. As shown in FIG. 4D, indexed beads with single indexingwas completed in the extension-ligation step of FIG. 4C. The solidsupport is contacted with an indexing mix, and the nucleic acidfragments are tagged for indexing. FIG. 4E depicts exemplary results ofthe various configurations, showing normalized read frequencies as afunction of insert size. FIG. 4E depicts normalized read frequency oftagged nucleic acid fragments from indexed or universal beads withsingle or dual indexing. FIGS. 4A-4E in particular depict embodiments ofthe transposome complexes arranged as indexed beads for single indexing(top left), universal beads for single indexing (top right), indexedbeads for dual indexing (bottom left), and universal beads for dualindexing. For the embodiments of indexed beads shown in FIGS. 4A-4E, forboth single and dual indexing (left), the attachment polynucleotide ishybridized to the second transposon at the 3′ adaptor sequence (B15′sequence). For the embodiments of universal beads shown in FIGS. 4A-4E,for both single and dual indexing (right), the attachment polynucleotideis hybridized to the first transposon at the 5′ adaptor sequence (A14sequence). In some embodiments, normalized libraries can be preparedfrom raw samples, such that a nucleic acid is extracted from a rawsample and directly inputted into a system or method described herein,where a self-normalized sample provides a tight CV across a range ofsample types.

FIGS. 5A and 5B depict a schematic diagram of exemplary transposomecomplexes for non-indexing, showing the attachment polynucleotidehybridized to either the P7 sequence-containing transposon (FIG. 5A) orthe P5 sequence-containing transposon (FIG. 5B). Specifically, FIGS. 5Aand 5B depicts a primer sequence (P5 sequence) joined to a 5′ adaptorsequence (A14 sequence) on the first transposon, and a primer sequence(P7′ sequence) joined to a 3′ adaptor sequence (B15′ sequence) on thesecond transposon, thereby providing non-indexing transposome complexes.The attachment polynucleotide could be hybridized on either the secondtransposon using a 5′ binding element (B) (FIG. 5A) or the firsttransposon using a 3′ binding element (B) (FIG. 5B).

FIGS. 6A-6D depict schematic diagrams of exemplary transposome complexesincluding an i5 indexing sequence. FIG. 6A depicts a complex in which anattachment polynucleotide comprising a nitro sequence and an anchorsequence can hybridize to an indexing oligonucleotide, which can then beligated to the 5′ end of the first transposon. In FIG. 6A, thetransposome complex comprises a first transposon comprising a 3′transposon end sequence (ME) and a 5′ adaptor sequence (A14), a secondtransposon comprising a 5′ transposon end sequence (ME′) and a 3′adaptor sequence (B15′), and an attachment polynucleotide comprising anattachment adaptor sequence (A14′) hybridized to the A14 in the firsttransposon, and a binding element (biotin). In this case, the attachmentpolynucleotide further comprises a nitroindole sequence (a universalsequence that binds to any i5 index region) and a primer sequence (P5′).The B15′ region of the second transposon can be hybridized to apolynucleotide comprising the complement (B15), an index region, and aP7 primer sequence, which itself is annealed to a P7′-i7′ indexmolecule. Ligation of the 5′ end of the i7′ index region to the 3′ endof the 3′ adaptor sequence serves to generate a fully double-strandedregion.

FIG. 6B depicts an attachment polynucleotide comprising an anchorsequence and a spacer region that can hybridize to an indexingoligonucleotide that can then be ligated to the 5′ end of the firsttransposon. In FIG. 6B, the first transposon comprises a 3′ transposonend sequence (ME) and a 5′ adaptor sequence (A14), and the secondtransposon comprises a 5′ transposon end sequence (ME′) and a 3′ adaptorsequence (B15′) as in FIG. 6A. In this case, though, the attachmentpolynucleotide comprises an adaptor complement (A14′), an anchor′sequence, a 2×sp 18 spacer region, a primer complement (P5′), and abiotin binding element. An i5 index comprises an anchor sequence(complementary to anchor′), an i5 index region, and a primer sequence(P5). The i5 index hybridizes to the complementary anchor and primersequences on the attachment polynucleotide across the spacer.

FIG. 6C depicts an attachment polynucleotide comprising a spacer and anA14′ sequence that can hybridize to an indexing oligonucleotide, whichcan then be ligated to sequence X at the 5′ end of the first transposon.In FIG. 6C, the first transposon comprises a 3′ transposon end sequence(ME) and a 5′ adaptor sequence X and the second transposon comprises a5′ transposon end sequence (ME′) and a 3′ adaptor sequence (B15′). Theattachment polynucleotide comprises the complement to the 5′ adaptorsequence (X′), a second adaptor sequence (A14′), a spacer region(2×sp18), a primer complement (P5′), and a biotin binding element. The5′ adaptor sequence (X′) hybridizes to the first transposon 5′ adaptorsequence (X). The i5 index comprises the complement to the secondadaptor sequence (A14), a 2×sp18 spacer region, and the primer sequence(P5). The i5 index hybridizes to the second adaptor sequence and primercomplement on the attachment polynucleotide, across the spacer region,and the 3′ end of the complement to the second adaptor sequence isligated to sequence X. In addition, an i7 index comprising a 5′ primersequence (P7), an i7 index region, and the complement to the 3′ adaptorsequence (B15) is annealed, and the 3′ end of the 3′ adaptor sequenceextended to produce a double-stranded region.

FIG. 6D depicts an attachment polynucleotide comprising a spacer and anA14′ sequence that can hybridize to an indexing oligonucleotide, whichcan then be ligated to sequence X at the 5′ end of the first transposon.In this case, the indexing oligonucleotide comprises a double-strandedprimer region. In FIG. 6D, the first transposon, second transposon, andattachment polynucleotide are as in FIG. 6C. The i7 index comprises adouble-stranded primer (P7/P7′), an i7 index region, and the complementto the 3′ adaptor sequence (B15). The double stranded region in the i7index can be created (annealed) during the extension ligation reaction,e.g., there is no need for the i7 index to be annealed prior to thereaction itself. The P7′ oligo can be included in the reaction mix. Thei7 index is annealed via the B15 region, and extension and ligation fromthe second transposon creates the double-stranded region. An example ofthis method is described in Example 5.

FIGS. 7A-7B depict exemplary results for performing a method ofgenerating a library of tagged nucleic acid fragments with and withoutusing PCR amplification, including results for improved indel precisionand recall (FIG. 7A) and improvement in coverage in GC rich promoters(FIG. 7B). Four methods were used to generate these data, two with PCR(TruSeq™ Nano and Nextera™ DNA Flex) and two PCR-free (the presentmethod and TruSeq™ PCR-Free). There were two replicates per method, witheight libraries generated in total. Data were down-sampled to 25× aftersequencing. FIG. 7A shows high percentage of indel precision and recallfor the PCR-free method described herein. From left to right, thesamples include (in duplicate): TruSeq™ Nano (black); Nextera™ DNA Flex(white); the present PCR-free method described herein (lined); andTruSeq™ PCR-Free (checked). FIG. 7B shows an improvement in coverage inGC rich promoters for the present PCR-free method described herein ascompared to other methods, including: Nextera™ DNA Flex (top left);TruSeq™ Nano (top right); the present PCR-free method described herein(bottom left); and TruSeq™ PCR-Free (bottom right).

FIG. 8 depicts results of sequencing libraries from a sample containinga known 100% GC repeat expansion (FMR1) prepared using Nextera™ DNA Flexor TruSeq™ DNA PCR-Free Library Prep Kits for comparison to the methodsdescribed herein (in quadruplicate Samples 1-4) as described in Example3.

FIGS. 9A-9C depict the % CV results for libraries prepared using eightindex pairs along with the systems of FIGS. 6C (data in FIG. 9A) and 6D(data for two reaction conditions in FIGS. 9B and 9C) as described inExample 5.

FIG. 10 depicts a graphical representation of sequencing coverage with agap in a region of gene RNPEPL1 for PCR library preparation methods(Nextera Flex or tube-based Nextera), but with fewer gaps using PCR freemethods described herein (two bottom panels).

DETAILED DESCRIPTION

Libraries of fragmented nucleic acids are often created from genomicnucleic acids for use in next generation sequencing (NGS) applications.The present disclosure provides for methods, compositions, and kits forgenerating a library of fragmented nucleic acids that appends sequencesneeded to perform sequencing operations, including indexes, withoutusing PCR to add the sequences by amplification (also referred to hereinas PCR-free library generation or PCR-free library preparation). ThisPCR-free transposome library preparation method may reduce and/oreliminate bias caused by PCR in current tagmentation approaches forlibrary preparation.

Tagmentation refers to the use of transposase to fragment and tagnucleic acids. Tagmentation includes the modification of DNA by atransposome complex comprising transposase enzyme complexed withadaptors comprising transposon end sequences (referred to herein astransposons). Tagmentation results in the simultaneous fragmentation ofthe DNA and ligation of the adaptors to the 5′ ends of both strands ofduplex fragments. Generally, following a purification step to remove thetransposase enzyme, additional sequences are added to the ends of theadapted fragments by PCR.

The methods, compositions, systems, and kits described herein relate tocomplex hybridized oligonucleotides, and transposome complexescomprising those hybrids, including complexes that are immobilized on asurface, and the use of the transposome complexes for PCR-free librarygeneration. As described herein, the transposome complex includes atransposase and associated transposons that fragment and tag a targetDNA molecule. In some aspects, the complex hybridized oligonucleotide iscleavable and includes a binding element, and in other aspects, thecomplex hybridized oligonucleotide comprises an attachmentpolynucleotide with a binding element. In some aspects, the attachmentpolynucleotide is a nucleic acid sequence that hybridizes to atransposon in a transposome complex and that is immobilized on a solidsupport, such as a slide, flow cell, or bead. Due to the hybridizationof the attachment polynucleotide to a transposon, the transposomecomplex may be immobilized on a solid support indirectly through theattachment polynucleotide. Binding of the attachment polynucleotide tothe solid support takes place through a binding element on theattachment polynucleotide. Target nucleic acids are captured by thetransposome complexes and the nucleic acids are then fragmented andtagged (“tagmentation”). The oligonucleotide system is designed to allowfor incorporation of any tags needed for indexing and sequencing viatagmentation, extension, and/or ligation steps, without PCRamplification. Thus, in some aspects, tagged fragments may be extendedand ligated, and indexed without amplification, to generate a library ofnucleic acid fragments without using PCR amplification.

Solution-based tagmentation has drawbacks and requires severallabor-intensive steps. Additionally, bias can be introduced during PCRamplification steps used to introduce tag sequences. For example,reduced indel may be a result of PCR due to polymerase slippage.Further, PCR polymerases have difficulty in some regions, such as highGC regions or AT or other sequence repeat regions, which leads to gapsor false structural variance calls in a genome, or missed repeatexpansions.

The methods, compositions, systems, and kits presented herein overcomethose drawbacks and allow unbiased sample preparation and sequencing tooccur with minimal requirements for sample manipulation or transfer. Themethods, compositions, systems, and kits described herein relate togenerating libraries without the use of PCR amplification. The PCR-freeapproach reduces and/or eliminates biases caused by PCR, including:reducing the number and frequency of gaps, particularly in GC richregions that are difficult to PCR; improving indel calling performance,including improving indel recall and indel precision; improving callingof repeat expansions; and improving coverage in GC rich promoters. Thepresent application discloses various transposome complex designs forperforming PCR-free tagmentation for improvement of generating nucleicacid libraries.

Furthermore, the methods, compositions, systems, and kits describedherein may be performed in a period of time less than nucleic acidsample preparation and analysis using other methods, such as PCR basedmethods. Thus, in some embodiments, the methods of generating a libraryof tagged nucleic acid fragments as described herein may be performed ina period of time of less than about 5 hours, for example, less than 5,less than 4, less than 3, or less than 2 hours. In some embodiments, themethods of generating a library of tagged nucleic acid fragments asdescribed herein may be performed in a period of time ranging from about90 minutes to about 300 minutes, such as 90, 105, 120, 135, 150, 165,180, 195, 210, 225, 240, 255, 270, 285, or 300 minutes, or for an amountof time within a range defined by any two of the aforementioned values.

Furthermore, in some embodiments, use of the methods, compositions,systems, and kits described herein results in fragmentation of nucleicacids that is not time dependent, immobilization of the transposomeresults in consistent insert size, and saturation allows for integratedextraction and a quantification-free library preparation.

Additional advantages of the methods, compositions, systems, and kitsdescribed herein relate to immobilization of the transposome complex ona solid surface, and include, for example reducing hands-on and overalllibrary preparation time, cost, and reagent requirements, loweringsample input requirements, and enabling the use of unpurified ordegraded samples as a starting point for library preparation. Inaddition, the transposome complexes described herein also producelibraries with more consistent insert sizes relative to solution-phasemethods, even when varying sample input concentrations are used.

In some embodiments, the nucleic acid libraries obtained by the methodsdisclosed herein can be sequenced using any suitable nucleic acidsequencing platform to determine the nucleic acid sequence of the targetsequence. In some respects, sequences of interest are correlated with orassociated with one or more congenital or inherited disorders,pathogenicity, antibiotic resistance, or genetic modifications.Sequencing may be used to determine the nucleic acid sequence of a shorttandem repeat, single nucleotide polymorphism, gene, exon, codingregion, exome, or portion thereof. As such, the methods and compositionsdescribed herein relate to creating sequenceable libraries useful in,but not limited to, cancer and disease diagnosis, prognosis andtherapeutics, DNA fingerprinting applications (e.g., DNA databanking,criminal casework), metagenomic research and discovery, agrigenomicapplications, and pathogen identification and monitoring.

In some embodiments, the attachment adaptor sequence is hybridized to atleast a portion of the 5′ adaptor sequence, and the binding element isat the 3′ end of the attachment polynucleotide. In some embodiments, theattachment adaptor sequence is hybridized to at least a portion of the3′ adaptor sequence, and the binding element is at the 5′ end of theattachment oligonucleotide.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. All patents, applications, published applications and otherpublications referenced herein are incorporated by reference in theirentirety unless stated otherwise. In the event that there are aplurality of definitions for a term herein, those in this sectionprevail unless stated otherwise. As used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Unlessotherwise indicated, conventional methods of mass spectroscopy, NMR,HPLC, protein chemistry, biochemistry, recombinant DNA techniques andpharmacology are employed. The use of “or” or “and” means “and/or”unless stated otherwise. Furthermore, use of the term “including” aswell as other forms, such as “include”, “includes,” and “included,” isnot limiting. As used in this specification, whether in a transitionalphrase or in the body of the claim, the terms “comprise(s)” and“comprising” are to be interpreted as having an open-ended meaning. Thatis, the terms are to be interpreted synonymously with the phrases“having at least” or “including at least.” When used in the context of aprocess, the term “comprising” means that the process includes at leastthe recited steps, but may include additional steps. When used in thecontext of a compound, composition, or device, the term “comprising”means that the compound, composition, or device includes at least therecited features or components, but may also include additional featuresor components.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Transposome Complexes

Some embodiments provided herein relate to a composition for generatinga library of tagged nucleic acid fragments without PCR amplification. Insome embodiments, the composition includes a solid support and atransposome complex immobilized to the solid support. In someembodiments, the transposome complex includes a transposase, a firsttransposon, an attachment polynucleotide, and a second transposon. Insome embodiments, the first transposon includes a 3′ transposon endsequence and a 5′ adaptor sequence. In some embodiments, the attachmentpolynucleotide includes an attachment adaptor sequence hybridized to the5′ adaptor sequence and a binding element. In some embodiments, thesecond transposon comprises a 5′ transposon end sequence and a 3′adaptor sequence. In some embodiments, the transposome complex isimmobilized to the solid support through the attachment polynucleotide.In some embodiments, the attachment polynucleotide further comprises aprimer sequence.

In some embodiments, the binding element comprises or is an optionallysubstituted biotin. In some embodiments, the binding element isconnected to the attachment polynucleotide via a linker. In someembodiments, the binding element comprises or is a biotin linker. Insome embodiments, the binding element comprises or is a 3′, 5′, orinternal biotin.

In some embodiments, the 3′ transposon end sequence comprises a mosaicend (ME) sequence and the 5′ transposon end sequence comprises an ME′sequence. In some embodiments, the 5′ adaptor sequence comprises an A14sequence and the attachment adaptor sequence comprises an A14′ sequence.In some embodiments, the 3′ adaptor sequence comprises a B15′ sequence.In some embodiments, the 3′ adaptor sequence is complementary to atleast a portion of an index adaptor sequence. In some embodiments, theindex adaptor sequence comprises a B15 sequence. In some embodiments, aportion of the attachment polynucleotide comprises a primer sequence,such as a P5′ primer sequence. In some embodiments, the primer sequenceof the attachment polynucleotide is complementary to at least a portionof an indexing oligonucleotide sequence, such as a P5 primer sequence.

In some embodiments, the transposome complex is immobilized on the solidsupport via the binding element (and optional linker) as describedherein. In some embodiments, the solid support is a bead, a paramagneticbead, a flowcell, a surface of a microfluidic device, a tube, a well ofa plate, a slide, a patterned surface, or a microparticle. In someembodiments, the solid support comprises or is a bead. In oneembodiment, the bead is a paramagnetic bead. In some embodiments, thesolid support comprises a plurality of solid supports. In someembodiments, transposome complexes are immobilized on a plurality ofsolid supports. In some embodiments, the plurality of solid supportscomprises a plurality of beads. In some embodiments, the plurality oftransposome complexes are immobilized on the solid support at a densityof at least 10³, 10⁴, 10⁵, 10⁶ complexes per mm². In some embodiments,the solid support is a bead or a paramagnetic bead, and there aregreater than 10,000, 20,000, 30,000, 40,000, 50,000, or 60,000transposome complexes bound to each bead.

Transposon based technology can be utilized for fragmenting DNA, forexample, as exemplified in the workflow for NEXTERA™ FLEX DNA samplepreparation kits (Illumina, Inc.), wherein target nucleic acids, such asgenomic DNA, are treated with transposome complexes that simultaneouslyfragment and tag (“tagmentation”) the target, thereby creating apopulation of fragmented nucleic acid molecules tagged with uniqueadaptor sequences at the ends of the fragments.

A transposition reaction is a reaction wherein one or more transposonsare inserted into target nucleic acids at random sites or almost randomsites. Components in a transposition reaction include a transposase (orother enzyme capable of fragmenting and tagging a nucleic acid asdescribed herein, such as an integrase) and a transposon element thatincludes a double-stranded transposon end sequence that binds to thetransposase (or other enzyme as described herein), and an adaptorsequence attached to one of the two transposon end sequences. One strandof the double-stranded transposon end sequence is transferred to onestrand of the target nucleic acid and the complementary transposon endsequence strand is not (a non-transferred transposon sequence). Theadaptor sequence can include one or more functional sequences orcomponents (e.g., primer sequences, anchor sequences, universalsequences, spacer regions, or index tag sequences) as needed or desired.

A “transposome complex” is comprised of at least one transposase (orother enzyme as described herein) and a transposon recognition sequence.In some such systems, the transposase binds to a transposon recognitionsequence to form a functional complex that is capable of catalyzing atransposition reaction. In some aspects, the transposon recognitionsequence is a double-stranded transposon end sequence. The transposasebinds to a transposase recognition site in a target nucleic acid andinserts the transposon recognition sequence into a target nucleic acid.In some such insertion events, one strand of the transposon recognitionsequence (or end sequence) is transferred into the target nucleic acid,resulting in a cleavage event. Exemplary transposition procedures andsystems that can be readily adapted for use with the transposases.

Exemplary transposases that can be used with certain embodimentsprovided herein include (or are encoded by): Tn5 transposase, SleepingBeauty (SB) transposase, Vibrio harveyi, MuA transposase and a Mutransposase recognition site comprising R1 and R2 end sequences,Staphylococcus aureus Tn552, Ty1, Tn7 transposase, Tn/O and IS10,Mariner transposase, Tc1, P Element, Tn3, bacterial insertion sequences,retroviruses, and retrotransposon of yeast. More examples include IS5,Tn10, Tn903, IS911, and engineered versions of transposase familyenzymes. The methods described herein could also include combinations oftransposases, and not just a single transposase.

In some embodiments, the transposase is a Tn5, Tn7, MuA, or Vibrioharveyi transposase, or an active mutant thereof. In other embodiments,the transposase is a Tn5 transposase or a mutant thereof. In otherembodiments, the transposase is a Tn5 transposase or a mutant thereof.In other embodiments, the transposase is a Tn5 transposase or an activemutant thereof. In some embodiments, the Tn5 transposase is ahyperactive Tn5 transposase, or an active mutant thereof. In someaspects, the Tn5 transposase is a Tn5 transposase as described in PCTPubl. No. WO2015/160895, which is incorporated herein by reference. Insome aspects, the Tn5 transposase is a hyperactive Tn5 with mutations atpositions 54, 56, 372, 212, 214, 251, and 338 relative to wild-type Tn5transposase. In some aspects, the Tn5 transposase is a hyperactive Tn5with the following mutations relative to wild-type Tn5 transposase:E54K, M56A, L372P, K212R, P214R, G251R, and A338V. In some embodiments,the Tn5 transposase is a fusion protein. In some embodiments, the Tn5transposase fusion protein comprises a fused elongation factor Ts (Tsf)tag. In some embodiments, the Tn5 transposase is a hyperactive Tn5transposase comprising mutations at amino acids 54, 56, and 372 relativeto the wild type sequence. In some embodiments, the hyperactive Tn5transposase is a fusion protein, optionally wherein the fused protein iselongation factor Ts (Tsf). In some embodiments, the recognition site isa Tn5-type transposase recognition site (Goryshin and Reznikoff, J.Biol. Chem., 273:7367, 1998). In one embodiment, a transposaserecognition site that forms a complex with a hyperactive Tn5 transposaseis used (e.g., EZ-Tn5™ Transposase, Epicentre Biotechnologies, Madison,Wis.). In some embodiments, the Tn5 transposase is a wild-type Tn5transposase.

In some embodiments, the transposome complex comprises a dimer of twomolecules of a transposase. In some embodiments, the transposome complexis a homodimer, wherein two molecules of a transposase are each bound tofirst and second transposons of the same type (e.g., the sequences ofthe two transposons bound to each monomer are the same, forming a“homodimer”). In some embodiments, the compositions and methodsdescribed herein employ two populations of transposome complexes. Insome embodiments, the transposases in each population are the same. Insome embodiments, the transposome complexes in each population arehomodimers, wherein the first population has a first adaptor sequence ineach monomer and the second population has a different adaptor sequencein each monomer.

In some embodiments, the transposase complex comprises a transposase(e.g., a Tn5 transposase) dimer comprising a first and a second monomer.In some aspects, each monomer comprises a first transposon, a secondtransposon, and an attachment polynucleotide, where the first transposonincludes a transposon end sequence at its 3′ end (also referred to as a3′ transposon end sequence) and an adaptor sequence at its 5′ end (alsoreferred to as a 5′ adaptor sequence); the second transposon includes atransposon end sequence at its 5′ end (also referred to as a 5′transposon end sequence) and an adaptor sequence at its 3′ end (alsoreferred to as a 3′ adaptor sequence); and the attachment polynucleotideincludes an attachment adaptor sequence hybridized to the 5′ adaptorsequence of the first transposon, a primer sequence, and a linker. Insome embodiments, the 5′ transposon end sequence of the secondtransposon is at least partially complementary to the 3′ transposon endsequence of the first transposon. In some embodiments, the attachmentadaptor sequence of the attachment polynucleotide is at least partiallycomplementary to the 5′ adaptor sequence of the first transposon. Insome embodiments, the linker of the attachment polynucleotide includes abinding element.

End Sequences

In any of the embodiments of the method described herein, the firsttransposon includes a 3′ transposon end sequence and the secondtransposon includes a 5′ transposon end sequence. In some embodiments,the 5′ transposon end sequence is at least partially complementary tothe 3′ transposon end sequence. In some embodiments, the complementarytransposon end sequences hybridize to form a double-stranded transposonend sequence that binds to the transposase (or other enzyme as describedherein). In some embodiments, the transposon end sequence is a mosaicend (ME) sequence. Thus, in some embodiments, the 3′ transposon endsequence is an ME sequence and the 5′ transposon end sequence is an ME′sequence.

Adaptor Sequences

In any of the embodiments of the method described herein, the firsttransposon includes a 5′ adaptor sequence and the second transposonincludes a 3′ adaptor sequence. Adaptor sequences may comprise one ormore functional sequences or components selected from the groupconsisting of primer sequences, anchor sequences, universal sequences,spacer regions, index sequences, capture sequences, barcode sequences,cleavage sequences, sequencing-related sequences, and combinationsthereof. In some embodiments, an adaptor sequence comprises a primersequence. In other embodiments, an adaptor sequence comprises a primersequence and an index or barcode sequence. A primer sequence may also bea universal sequence. This disclosure is not limited to the type ofadaptor sequences that could be used and a skilled artisan willrecognize additional sequences that may be of use for librarypreparation and next generation sequencing. A universal sequence is aregion of nucleotide sequence that is common to two or more nucleic acidfragments. Optionally, the two or more nucleic acid fragments also haveregions of sequence differences. A universal sequence that may bepresent in different members of a plurality of nucleic acid fragmentscan allow for the replication or amplification of multiple differentsequences using a single universal primer that is complementary to theuniversal sequence.

In some embodiments, the attachment polynucleotide includes anattachment adaptor sequence hybridized to the 5′ adaptor sequence. Insome embodiments, the attachment adaptor sequence is at least partiallycomplementary to the 5′ adaptor sequence. In some embodiments, theadaptor sequence is an A14 sequence or a B15 sequence. Thus, in someembodiments, the 5′ adaptor sequence is an A14 sequence and theattachment adaptor sequence is an A14′ sequence. In some embodiments,the 3′ adaptor sequence is a B15′ sequence. In some embodiments, theadaptor sequence is any sequence for hybridization (referred to hereinas sequence X). In some embodiments, sequence X comprises 16-20nucleotides. In some embodiments, sequence X has a similar meltingtemperature (Tm) to an adapter sequence. In some embodiments, sequencingresults are improved when the Tm of sequence X has a similar meltingtemperature to that of an adapter sequence. In some embodiments,sequence X has a similar melting temperature to an A14 sequence or B15sequence. In some embodiments, the Tm of sequence X is 53°-56°. In someembodiments, adaptor sequences are transferred to the 5′ ends of anucleic acid fragment by a tagmentation reaction.

In any of the embodiments, the adaptor sequence or transposon endsequences, including A14-ME, ME, B15-ME, ME′, A14, B15, and ME areprovided below:

A14-ME: 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′ (SEQ ID NO: 1)

B15-ME: 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′ (SEQ ID NO: 2)

ME′: 5′-phos-CTGTCTCTTATACACATCT-3′ (SEQ ID NO: 3)

A14: 5′-TCGTCGGCAGCGTC-3′ (SEQ ID NO: 4)

B15: 5′-GTCTCGTGGGCTCGG-3′ (SEQ ID NO: 5)

ME: AGATGTGTATAAGAGACAG (SEQ ID NO: 6)

Attachment Polynucleotide

Embodiments of the transposome complex described herein include anattachment polynucleotide. As used herein, the attachment polynucleotideis a polynucleotide that hybridizes to a transposon on one end and bindsto a surface on a second end. Thus, the transposome complex describedherein is immobilized to a solid support through the attachmentpolynucleotide. In some embodiments, an attachment polynucleotideincludes an attachment adaptor sequence hybridized to the adaptorsequence of the first transposon or the adaptor sequence of the secondtransposon, a primer sequence, and a linker. In some embodiments, thelinker includes a binding element.

As described herein the attachment adaptor sequence may be at leastpartially complementary to the adaptor sequence of the first or secondtransposon. In some embodiments, the attachment adaptor sequencehybridizes to the 5′ adaptor sequence. In embodiments when theattachment adaptor sequence hybridizes to the 5′ adaptor sequence, wherethe 5′ adaptor sequence is an A14 sequence, the attachment adaptorsequence is an A14′ sequence. In some embodiments, the adaptor sequenceis sequence X. In some embodiments, the attachment adaptor sequencehybridizes to the 3′ adaptor sequence. In embodiments when theattachment adaptor sequence hybridizes to the 3′ adaptor sequence, wherethe 3′ adaptor sequence is a B15′ sequence, the attachment adaptorsequence is a B15 sequence. In any of these embodiments, the attachmentadaptor sequence may be fully complementary to the adaptor sequence ofthe first or second transposon or partially complementary to the adaptorsequence of the first or second transposon.

In some embodiments, the attachment polynucleotide contains a primersequence. In some embodiments, the primer sequence is a P5 primersequence or a P7 primer sequence or a complement thereof (e.g., P5′ orP7′). The P5 and P7 primers are used on the surface of commercial flowcells sold by Illumina, Inc., for sequencing on various Illuminaplatforms. The primer sequences are described in U.S. Pat. Publ. No.2011/0059865, which is incorporated herein by reference in its entirety.Examples of P5 and P7 primers, which may be alkyne terminated at the 5′end, include the following:

P5: AATGATACGGCGACCACCGAGAUCTACAC (SEQ ID NO: 7)

P7: CAAGCAGAAGACGGCATACGAG*AT (SEQ ID NO: 8)

and derivatives thereof. In some examples, the P7 sequence includes amodified guanine at the G* position, e.g., an 8-oxo-guanine. In otherexamples, the * indicates that the bond between the G* and the adjacent3′ A is a phosphorothioate bond. In some examples, the P5 and/or P7primers include unnatural linkers. Optionally, one or both of the P5 andP7 primers can include a poly T tail. The poly T tail is generallylocated at the 5′ end of the sequence shown above, e.g., between the 5′base and a terminal alkyne unit, but in some cases can be located at the3′ end. The poly T sequence can include any number of T nucleotides, forexample, from 2 to 20. While the P5 and P7 primers are given asexamples, it is to be understood that any suitable primers can be usedin the examples presented herein. The index sequences having the primersequences, including the P5 and P7 primer sequences serve to add P5 andP7 for activating the library for sequencing. While the P5 and P7primers are given as examples, it is to be understood that any suitableamplification primers can be used in the examples presented herein.

As used herein, one example of a linker is a moiety that covalentlyconnects a binding element to the end of the nucleotide portion of theattachment polynucleotide and may be used to immobilize the attachmentpolynucleotide to a solid support. The linker may be a cleavable linker,for example, a linker capable of being cleaved to remove the attachmentpolynucleotide, and thus the transposome complex or tagmentation productfrom the solid support. A cleavable linker as used herein is a linkerthat may be cleaved through chemical or physical means, such as, forexample, photolysis, chemical cleavage, thermal cleavage, or enzymaticcleavage. In some embodiments the cleavage may be by biochemical,chemical, enzymatic, nucleophilic, reduction sensitive agent or othermeans. Cleavable linkers may comprise a moiety selected from the groupconsisting of: a restriction endonuclease site; at least oneribonucleotide cleavable with an RNAse; nucleotide analogues cleavablein the presence of certain chemical agent(s); photo-cleavable linkerunit; a diol linkage cleavable by treatment with periodate (forexample); a disulfide group cleavable with a chemical reducing agent; acleavable moiety that may be subject to photochemical cleavage; and apeptide cleavable by a peptidase enzyme or other suitable means.Cleavage may be mediated enzymatically by incorporation of a cleavablenucleotide or nucleobase into the cleavable linker, such as uracil or8-oxo-guanine.

In some embodiments, the linker described herein may be covalently anddirectly attached the attachment polynucleotide, for example, forming a—O— linkage, or may be covalently attached through another group, suchas a phosphate or an ester. Alternatively, the linker described hereinmay be covalently attached to a phosphate group of the attachmentpolynucleotide, for example, covalently attached to the 3′ hydroxyl viaa phosphate group, thus forming a —O—P(O)₃— linkage.

A binding element, as used herein, is a moiety that can be used to bind,covalently or non-covalently, to a binding partner. In some aspects, thebinding element is on the transposome complex and the binding partner ison the solid support. In some embodiments, the binding element can bindor is bound non-covalently to the binding partner on the solid support,thereby non-covalently attaching the transposome complex to the solidsupport. In some embodiments, the binding element is capable of binding(covalently or non-covalently) to a binding partner on a solid support.In some aspects, the binding element is bound (covalently ornon-covalently) to a binding partner on the solid support, resulting inan immobilized transposome complex.

In such embodiments, the binding element comprises or is, for example,biotin, and the binding partner comprises or is avidin or streptavidin.In other embodiments, the binding element/binding partner combinationcomprises or is FITC/anti-FITC, digoxigenin/digoxigenin antibody, orhapten/antibody. Further suitable binding pairs include, but not limitedto, dithiobiotin-avidin, iminobiotin-avidin, biotin-avidin,dithiobiotin-succinilated avidin, iminobiotin-succinilated avidin,biotin-streptavidin, and biotin-succinilated avidin. In someembodiments, the binding element is a biotin and the binding partner isstreptavidin.

In some embodiments, the binding element can bind to the binding partnervia a chemical reaction or is bound covalently by reaction with thebinding partner on the solid support, thereby covalently attaching thetransposome complex to the solid support. In some aspects, the bindingelement/binding partner combination comprises or is amine/carboxylicacid (e.g., binding via standard peptide coupling reaction underconditions known to one of ordinary skill in the art, such as EDC orNETS-mediated coupling). The reaction of the two components joins thebinding element and binding partner through an amide bond.Alternatively, the binding element and binding partner can be two clickchemistry partners (e.g., azide/alkyne, which react to form a triazolelinkage).

In some embodiments, the attachment polynucleotide further includesadditional sequences or components, such as a universal sequence, aspacer region, an anchor sequence, or an index tag sequence, or acombination thereof. A universal sequence is a region of nucleotidesequence that is common to two or more nucleic acid fragments.Optionally, the two or more nucleic acid fragments also have regions ofsequence differences. A universal sequence that may be present indifferent members of a plurality of nucleic acid fragments can allow forthe replication or amplification of multiple different sequences using asingle universal primer that is complementary to the universal sequence.

In some embodiments, the first transposon further comprises a primersequence 5′ of the 5′ adaptor sequence, and the attachmentpolynucleotide comprises (i) a portion complementary with and hybridizedto the 5′ adaptor sequence and (ii) a complementary primer sequence(see, e.g., FIG. 4A, single index, universal beads). Such constructs areuseful as universal beads for single indexing applications as there isno index tag sequence employed.

In some embodiments, the first transposon further comprises a primersequence 5′ of the 5′ adaptor sequence, and the attachmentpolynucleotide comprises an index tag sequence and a primer sequence(see, e.g., FIG. 4A, single indexing, indexed beads).

In some embodiments, the first transposon comprises the 5′ adaptorsequence, and the attachment polynucleotide comprises (i) a portioncomplementary with and hybridized to the 5′ adaptor sequence, (ii) aspacer region, and (iii) a primer sequence (see, e.g., FIG. 4A,universal beads, dual indexing).

In some embodiments, the second transposon comprises the 3′ adaptorsequence and the attachment polynucleotide comprises (i) a portioncomplementary to and hybridized with the 3′ adaptor sequence, (ii) anindex tag sequence, and (iii) a primer sequence (see, e.g., FIG. 4A,dual indexing, indexed beads).

In some embodiments, the attachment polynucleotide comprises a spacerregion (see, e.g., FIG. 4A) or a spacer region and an anchor region(see, e.g., FIG. 3 ). As used herein, a spacer region is a sequencerefers a nucleic acid sequence not carrying any structural or codifyinginformation for known gene functions. The spacer region on theattachment polynucleotide is capable of aligning with indexingoligonucleotides with varied sequences (e.g., with a range of i5sequences). In some embodiments, the spacer region is a universalsequence. In some embodiments, the spacer region is a non-DNA spacer. Insome embodiments, the spacer region includes universal bases, such asinosines or nitroindoles. In some embodiments, the spacer includes asp18 linker. A sp18 linker, as used herein, is a standard modificationlinker having C18 spacers (an 18-atom hexa-ethylene glycol spacer), andis equivalent to 4 base pairs in length. Thus, a 2×sp18 linker isequivalent to 8 base pairs in length. In some embodiments, the spacerregion comprises a 2×sp18 synthetic linker. In some embodiments, thespacer region comprises one or more C18 spacers, such as 1, 2, 3, 4, 5,6, or more C18 spacers. In some embodiments, the spacer region comprisestwo C18 spacers (which are equivalent in length to 8 nucleotides). Insome embodiments, the spacer is a C9 spacer equivalent in length to 2base pairs. In some embodiments, the spacer region comprises one or moreC9 spacers (triethyleneglycol spacer), such as 1, 2, 3, 4, 5, 6, or moreC9 spacers. In some embodiments, the spacer is a conventional spacerused with existing indices, such as a 10 base pair spacer. In someembodiments, the spacer region is a combination of spacers, for example,a combination of one or more C18 spacers and one or more C9 spacers, orany combination of any spacer described herein. In some embodiments, thespacer region is a length equivalent to 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,15, 20, or 30 base pairs. In some embodiments, the spacer region is alength approximately equivalent to 8 or 10 base pairs or nucleotides. Insome embodiments, the spacer region is specifically chosen to be thesame length as the index region. In some embodiments, the index regionsare 8 nucleotides long, and the spacer region comprises two C18 spacers.In some embodiments, the index regions are 10 nucleotides long and thespacer region comprises two C18 spacers and one C9 spacer.

In some aspects, the attachment polynucleotide comprises an anchorsequence. In some embodiments, the anchor sequence is GGATATGCTCGG (SEQID NO: 22). In some embodiments, the anchor sequence is an A14 sequence(SEQ ID NO: 4). As used herein, an anchor region means a DNA sequencethat is complementary to an anchor complement region in an indexingoligonucleotide and enables hybridization of the two components (see,e.g., FIG. 3B). In some aspects, the anchor region is complementary to aportion of an anchor complement region of an indexing oligonucleotide,where the indexing oligonucleotide comprises the anchor regioncomplement and an index tag sequence (-Anchor′-Index Tag Sequence-). Insome embodiments, the anchor sequence is complementary to an anchorcomplement region common to a plurality of indexing oligonucleotides. Insome embodiments, each index tag sequence in a plurality of indexingoligonucleotides is the same (no indexing) or different (indexing). Insome embodiments, the index tag sequence is an i5 sequence. Theattachment polynucleotide may further include additional sequenceelements or components for improving efficiency and functionality of theattachment polynucleotide for binding to indices, including, forexample, primer sequences, anchor sequences, universal sequences, spacerregions, index sequences, capture sequences, barcode sequences, cleavagesequences, sequencing-related sequences, and combinations thereof. Insome embodiments, the attachment polynucleotide comprises an A14′sequence.

Variations of the transposome complex, including the transposase, thetransposons, and the attachment polynucleotide may be realized. Forexample, variations in configuration, design, hybridization, structuralelements, and overall arrangement of the transposome complex may berealized. The disclosure and drawings provided herein provide severalvariations, but it is understood that additional variations within thescope of the disclosure may be readily realized.

Solid Support

The terms “solid surface,” “solid support,” and other grammaticalequivalents refer to any material that is appropriate for or can bemodified to be appropriate for the attachment of the transposomecomplexes. As will be appreciated by those in the art, the number ofpossible substrates is multitude. Possible substrates include, but arenot limited to, glass and modified or functionalized glass, plastics(including acrylics, polystyrene and copolymers of styrene and othermaterials, polypropylene, polyethylene, polybutylene, polyurethanes,TEFLON, etc.), polysaccharides, polyhedral organic silsesquioxane (POSS)materials, nylon or nitrocellulose, ceramics, resins, silica, orsilica-based materials including silicon and modified silicon, carbon,metals, inorganic glasses, plastics, optical fiber bundles, beads,paramagnetic beads, and a variety of other polymers.

Suitable bead compositions include, but are not limited to, plastics,ceramics, glass, polystyrene, methylstyrene, acrylic polymers,paramagnetic materials, thoria sol, carbon graphite, titanium dioxide,latex or cross-linked dextran such as Sepharose, cellulose, nylon,cross-linked micelles and TEFLON, as well as any other materialsoutlined herein for solid supports. In certain embodiments, themicrospheres are magnetic microspheres or beads, for exampleparamagnetic particles, spheres or beads. The beads need not bespherical; irregular particles may be used. Alternatively oradditionally, the beads may be porous. The bead sizes range fromnanometers, e.g., 100 nm, to millimeters, e.g., 1 mm, with beads fromabout 0.2 micron to about 200 microns being preferred, and from about0.5 to about 5 micron being particularly preferred, although in someembodiments smaller or larger beads may be used. The bead may be coatedwith a binding partner, for example the bead may be streptavidin coated.In some embodiments, the beads are streptavidin coated paramagneticbeads, for example, Dynabeads MyOne streptavidin C1 beads (ThermoScientific catalog #65601), Streptavidin MagneSphere Paramagneticparticles (Promega catalog #Z5481), Streptavidin Magnetic beads (NEBcatalog #S1420S) and MaxBead Streptavidin (Abnova catalog #U0087). Thesolid support could also be a slide, for example a flowcell or otherslide that has been modified such that the transposome complex can beimmobilized thereon.

In some embodiments, the binding partner is present on the solid supportor bead at a density of from 1000 to about 6000 pmol/mg, or about 2000to about 5000 pmol/mg, or about 3000 to about 5000 pmol/mg, or about3500 to about 4500 pmol/mg.

In one embodiment, the solid surface is the inner surface of a sampletube. In another embodiment, the solid surface is a capture membrane. Inone example, the capture membrane is a biotin-capture membrane (forexample, available from Promega Corporation). In another example, thecapture membrane is filter paper. In some embodiments of the presentdisclosure, solid supports comprised of an inert substrate or matrix(e.g. glass slides, polymer beads etc.) which has been functionalized,for example by application of a layer or coating of an intermediatematerial comprising reactive groups which permit covalent attachment tomolecules, such as polynucleotides. Examples of such supports include,but are not limited to, polyacrylamide hydrogels supported on an inertsubstrate such as glass, particularly polyacrylamide hydrogels asdescribed in WO2005/065814 and US2008/0280773, the contents of which areincorporated herein in their entirety by reference. The methods oftagmenting (fragmenting and tagging) DNA on a solid surface for theconstruction of a tagmented DNA library are described in WO2016/189331and US2014/0093916A1, which are incorporated herein by reference intheir entireties. In some embodiments, the transposome complex describedherein is immobilized to a solid support via the binding element. Insome such embodiments, the solid support comprises streptavidin as thebinding partner and the binding element is biotin.

In some embodiments, transposome complexes are immobilized on a solidsupport, such as a bead, at a particular density or density range. Insome embodiments, the density of complexes on a solid support refers tothe concentration of transposome complexes in solution during theimmobilization reaction. The complex density assumes that theimmobilization reaction is quantitative. Once the complexes are formedat a particular density, that density remains constant for the batch ofsurface-bound transposome complexes. The resulting beads can be diluted,and the resulting concentration of complexes in the diluted solution isthe prepared density for the beads divided by the dilution factor.Diluted bead stocks retain the complex density from their preparation,but the complexes are present at a lower concentration in the dilutedsolution. The dilution step does not change the density of complexes onthe beads, and therefore affects library yield but not insert (fragment)size. In some embodiments, the density is between about 5 nM and about1000 nM, or between about 5 and 150 nM, or between about 10 nM and 800nM. In other embodiments, the density is about 10 nM, or about 25 nM, orabout 50 nM, or about 100 nM, or about 200 nM, or about 300 nM, or about400 nM, or about 500 nM, or about 600 nM, or about 700 nM, or about 800nM, or about 900 nM, or about 1000 nM. In some embodiments, the densityis about 100 nM. In some embodiments, the density is about 300 nM. Insome embodiments, the density is about 600 nM. In some embodiments, thedensity is about 800 nM. In some embodiments, the density is about 100nM. In some embodiments, the density is about 1000 nM.

Some embodiments provided herein relate to a kit including a transposomecomplex, as described herein, and an index mix comprising indexsequences. Some embodiments provided herein relate to a kit including acomposition having a solid support with a transposome compleximmobilized thereon, as described herein, and an index mix comprisingindex sequences. In some embodiments, the index mix comprises i5 indexsequences and i7 index sequences, wherein the i5 index sequences arecomplementary to and hybridize to the attachment polynucleotide, andwherein the i7 index sequences are complementary to and hybridize to the3′ adaptor sequence.

Immobilized Transposome Complexes/Methods of Generating Tagged NucleicAcid Fragments

The disclosure further provides methods of preparing immobilizedtransposome complexes as described herein. Some embodiments providedherein relate to a method of generating a library of tagged nucleic acidfragments. In some embodiments, the method includes providing a solidsupport having a transposome complex immobilized thereon, applying atarget nucleic acid to the solid support under conditions sufficient tofragment the target nucleic acid into a plurality of target fragments,and to join the 3′ end of the first transposon to the 5′ ends of thetarget fragments to produce a plurality of 5′ tagged target fragments,and applying an index mix comprising index sequences to activate thelibrary for sequencing. In some embodiments, the transposome complexincludes a transposase bound to a first and a second transposon, a firsttransposon comprising a 3′ transposon end sequence and a 5′ adaptorsequence, an attachment polynucleotide comprising an attachment adaptorsequence and a binding element, and a second transposon comprising a 5′transposon end sequence and a 3′ adaptor sequence.

In some embodiments, a method comprises generating a library of taggednucleic acid fragments comprising contacting an immobilized transposomecomplex with a target nucleic acid under conditions sufficient tofragment the target nucleic acid into a plurality of target fragments,and to join the 3′ end of the first transposon to the 5′ ends of thetarget fragments to produce a plurality of 5′ tagged target fragments.

In some aspects, the method further comprises: treating the solidsupport to remove unbound nucleic acids; or treating the solid supportto remove the transposase from the complex, optionally by (a) heatingthe solid support and/or (b) washing the solid support with an enzymedenaturing agent, wherein the enzyme denaturing agent optionallycomprises sodium dodecyl sulfate (SDS), guanidine hydrochloride, urea,or proteinase.

In some embodiments, the contacting comprises adding a biological sampleto the transposome complex. In some embodiments, the biological samplecomprises a cell lysate or whole cells, or is selected from blood,plasma, serum, lymph, mucus, sputum, urine, semen, cerebrospinal fluid,bronchial aspirate, feces, macerated tissue, and fixed tissue FFPE.

In some aspects, the contacting further comprises hybridizing aplurality of indexing oligonucleotides to the attachmentpolynucleotides, wherein the plurality of indexing oligonucleotidescomprise the same or different index sequences.

In some aspects the method further comprises treating the plurality of5′ tagged target fragments with a polymerase and a ligase to extend andligate the strands to produce fully double-stranded tagged fragments. Insome aspects, the treating with a polymerase and a ligase is done in thepresence of a DNA secondary structure disruptor, wherein the disruptoris optionally DMSO. In some aspects, the treating with a polymerase anda ligase is done in the presence of an oligonucleotide that is acomplement of the primer sequence, wherein the oligonucleotide is a P7′oligonucleotide, and wherein the primer sequence is a P7 sequence. Insome embodiments, the polymerase comprises a T4 DNA polymerase mutantlacking exonuclease activity.

In some aspects, the method further comprises removing the fullydouble-stranded tagged fragments from the solid support. In someaspects, the removing comprises applying heat and/or a denaturantsufficient to cleave the fully double-stranded tagged fragments from thesolid support.

In some aspects, the method further comprising sequencing one or more ofthe 5′ tagged target fragments or fully double-stranded taggedfragments. In some aspects, the fragments are not quantified by PCRafter the contacting and before the sequencing. In some aspects, thenucleic acid is DNA or RNA. In some aspects, the 3′ adaptor sequence iscomplementary to at least a portion of an index adaptor sequence. Insome aspects, the primer sequence of the attachment polynucleotide iscomplementary to at least a portion of an index primer sequence. In someaspects, the primer sequence of the attachment polynucleotide iscomplementary to an index primer sequence of the indexingpolynucleotide.

In some embodiments, the method further includes washing the solidsupport to remove unbound nucleic acids. In some embodiments, the methodfurther includes treating the solid support to remove transposase. Insome embodiments, treating the solid support includes washing the solidsupport with an enzyme denaturing agent. In some embodiments, the enzymedenaturing agent includes acetic acid, dimethyl sulfoxide (DMSO),dithiothreitol, ethanol, formaldehyde, formamide, glutaraldehyde,guanidine hydrochloride, lithium perchlorate, mercaptoethanol, propyleneglycol, proteinase, sodium bicarbonate, sodium dodecyl sulfate (SDS),sodium salicylate, sulfosalicylic acid, trichloroacetic acid,tris(2-carboxyethyl)phosphine) (TCEP), or urea. In some embodiments,treating the solid support includes heating the solid support to removetransposase. In some embodiments, applying a target nucleic acidincludes mixing a biological sample with the transposome complex. Thenucleic acid need not be completely purified or purified at all, and canbe part of a biological sample or a mixture with protein, other nucleicacid species, other cellular components, and/or any other contaminants.In some embodiments, the biological sample includes a cell lysate. Insome embodiments, the biological sample includes whole cells. In someembodiments, the biological sample is selected from a bodily fluid,blood, plasma, serum, lymph, mucus, sputum, urine, semen, cerebrospinalfluid, bronchial aspirate, feces, macerated tissue, and fixed tissueformalin-fixed paraffin-embedded tissue (FFPE).

In some embodiments, applying the index mix includes hybridizing anindex sequence to the attachment polynucleotide and to the secondtransposon. In some embodiments, the index mix comprises anextension-ligation mix (ELM). In some embodiments, the ELM comprises apolymerase and a ligase. In some embodiments, the ELM is a split ELM forseparation ligation of indices. In some embodiments, the polymerasecomprises T4 DNA polymerase or a mutant thereof (such as a T4 DNApolymerase lacking exonuclease activity, or a mutant T4 DNA polymerase,or a mutant T4 DNA polymerase lacking exonuclease activity). In someembodiments, the ligase comprises E. coli DNA ligase. In someembodiments, the extension-ligation reaction is done in the presence ofa DNA secondary structure disruptor, such as DMSO. In some embodiments,the method further includes denaturing the plurality of 5′ tagged targetfragments from the solid support. In some embodiments, denaturing may beachieved by applying heat and/or a denaturant sufficient to cleave the5′ tagged target fragments from the solid support. In some embodiments,the method further includes sequencing one or more of the 5′ taggedtarget fragments or ligation products thereof. In some embodiments, thenucleic acid is DNA or RNA.

In some aspects, such methods include contacting the first and secondtransposons as described herein with an attachment polynucleotide boundto a surface under conditions suitable for hybridizing the attachmentpolynucleotide to the first or second transposon. In some aspects,methods for preparing a solid support-bound transposome complex compriseincubating a transposome complex as described herein with a solidsupport comprising a binding partner under conditions sufficient for thebinding element to bind (covalently or non-covalently) with the bindingpartner. The immobilized hybridized polynucleotide that includes theattachment polynucleotide and the first and second transposons is thencontacted with transposase under conditions suitable for forming atransposome complex.

In some embodiments, the first and second transposons as describedherein are annealed to each other, and the first transposon is annealedto the attachment polynucleotide. The annealed polynucleotides are thenloaded onto a transposase, such as a Tn5 transposase, thereby forming atransposome complex, which is then contacted with and bound to a solidsupport, such as a bead. In some embodiments, the annealed transposonsare bound to a solid support such as a bead and a transposase is thencomplexed with the transposons, thereby creating a transposome that isbound to a solid support.

In some aspects, methods are provided for preparing fragments from atarget nucleic acid, the method comprising providing a solid supportcomprising a transposome complex immobilized thereon as describedherein; applying a target nucleic acid to the solid support underconditions sufficient to fragment the target nucleic acid into aplurality of target fragments, and to join the 3′ end of the firsttransposon to the 5′ ends of the target fragments to produce a pluralityof 5′ tagged target fragments. In some aspects, the method furtherincludes applying an index mix including index sequences. In someembodiments, the fragment condition is a condition suitable fortagmentation by using the transposome complex to fragment and tag thetarget nucleic acid.

In some embodiments of the methods described herein, following thefragmenting and tagging, the methods further include washing the solidsupport to remove unbound nucleic acids. In some embodiments of themethods described herein, following the fragmenting and tagging, themethods further include removing the transposase. Removal of thetransposase may be accomplished under chemical conditions, such as,washing the solid support with an agent to remove the transposase. Insome embodiments, the agent is sodium dodecyl sulfate (SDS).

In some embodiments, the method further includes contacting the solidsupport with an index mix. Contacting with an index mix serves to tagthe fragments with a particular index and to activate the library forsequencing. In some embodiments, the index mix includes oligonucleotidesthat hybridize to the transposon or the attachment polynucleotide to tagor index the nucleic acid fragments. Thus, for example, theoligonucleotide includes an index and other regions of theoligonucleotide are complementary to the transposon or attachmentpolynucleotide and hybridizes thereto. By way of example, in oneembodiment, an i5 index having a primer sequence (P5 sequence), an indexsequence (i5 sequence), and an anchor sequence hybridizes to theattachment polynucleotide at complementary sequences, such that P5hybridizes to P5′, and anchor hybridizes to anchor′. In someembodiments, the i5 sequence binds to a nitroindol (nitro) sequence ofthe attachment polynucleotide. In some embodiments, a nitroindolsequence is complementary to and hybridizes to any i5 index sequence. Inone embodiment, an i7 index having a primer sequence (P7), an indexsequence (i7), and an adaptor sequence (B15 sequence) hybridizes to thesecond transposon at complementary sequences, such that the P7hybridizes to P7′, the i7 hybridizes to i7′, and B15 hybridizes to B15′.In some embodiments, the index mix includes a double stranded index.After contacting the solid support with the index mix, the fragments areligated and extended using an extension and ligation mix (ELM). An ELMmay include, for example a T4 DNA polymerase and an E. coli DNA ligase.Exemplary polymerases include, but are not limited, to, the Bst largefragments of Bst DNA polymerase I, E. coli DNA polymerase I (Klenowfragment), Klenow fragment (3′-5′ exo-), T4 DNA polymerase, T7 DNApolymerase, Deep VentR. (exo-) DNA Polymerase, Deep VentR DNAPolymerase, Therminator II DNA Polymerase, AmpliTherm DNA Polymerase,SP6 DNA polymerase, or Taq polymerase, or mutants, analogues, orderivatives of any of the aforementioned polymerases. Exemplary ligasesinclude, but are not limited to T4 DNA ligase, T4 RNA ligase, Taq DNAligase, E. Coli DNA ligase, Pfu DNA ligase and Tth DNA ligase. In someembodiments, the ELM is a split ELM reaction to allow for index ligationand index extension of difference indices, for example, to allow forseparate i5 ligation and i7 extension. The solid support is then treatedwith an agent to denature the strand sequences, such as with NaOH,thereby generating tagged nucleic acid fragments.

In some embodiments, the fragments are deposited on a flow cell. In someembodiments, the fragments are hybridized to complementary primersgrafted to the flow cell or surface. In some embodiments, the sequencesof the sequencing fragments are detected by array sequencing ornext-generation sequencing methods, such as sequencing-by-synthesis.

Table 1 depicts exemplary sequences used in the transposome complexesfor generating the library of tagged nucleic acid fragments.

TABLE 1 Exemplary Sequences Oligo- nucleotide name Design FunctionSequence (SEQ ID NO) P5_A14_ME Single indexing, indexed5′ transposon arm AATGATACGGCGACCA and universal beads CCGAGATCTACACTCGTCGGCAGCGTCAGATG TGTATAAGAGACAG (SEQ ID NO: 9) P_ME′_ B15′All designs involving i7 3′ transposon arm /5Phos/CTGTCTCTTATA indexingCACATCTCCGAGCCCA CGAGAC (SEQ ID NO: 10) Bio_P7_i701_ i701 indexed beads,Attachment oligo /5Biosg/CAAGCAGAAGA B15_ddC single or dual indexingCGGCATACGAGATTCG CCTTAGTCTCGTGGGCT CGG/3ddC/ (SEQ ID NO: 11)A14′_P5′_bio Universal beads, single  Attachment oligo GACGCTGCCGACGAGTindexing GTAGATCTCGGTGGTC GCCGTATCATT/3Bio/ (SEQ ID NO: 12) P_Al4_ MESuitable for dual 5′ transposon arm /5Phos/TCGTCGGCAGCG indexingTCAGATGTGTATAAGA GACAG (SEQ ID NO: 13) A14′_nitro_ Nitroindole universalAttachment oligo GACGCTGCCGACGACC P5′_bio design C/i5NitInd//i5NitInd//i5NitInd//i5NitInd// i5NitInd//i5NitInd// i5NitInd//i5NitInd/GTGTAGATCTCGGTGGTC GCCGTATCATT/3Bio/ (SEQ ID NO: 14) A14′12anc′_2sp_Anchor and spacer Attachment oligo GACGCTGCCGACGACC p5′_biouniversal design GAGCATATCC/iSp18// iSp18/GTGTAGATCTCGGTGGTCGCCGTATCATT/ 3BioTEG/(SEQ ID NO: 15) P5_i501 For use with nitroindolei501 indexing oligo AATGATACGGCGACCA design CCGAGATCTACACTAGATCGC (SEQ ID NO: 16) P_i701′_P7′_ddC Part of double-stranded  i701 indexing oligo /5Phos/TAAGGCGAATCT i7 adaptor, can be usedCGTATGCCGTCTTCTGC with several bead  TTG/3ddC/ (SEQ ID NO: designs 17)P7_i701_B15_ddC Part of double-stranded i701 indexing oligoCAAGCAGAAGACGGCA i7 adaptor, can be used TACGAGATTCGCCTTAwith several bead  GTCTCGTGGGCTCGG/ designs 3ddC/ (SEQ ID NO: 18)P5_i501_12anc For use with anchor and i501 indexing oligoAATGATACGGCGACCA spacer attachment oligo CCGAGATCTACACTAGATCGCGGATATGCTCGG (SEQ ID NO: 19)

In some aspects, methods of generating a library of tagged nucleic acidfragments comprise contacting an immobilized transposome complex with atarget nucleic acid under conditions sufficient to fragment the targetnucleic acid into a plurality of target fragments, and to join the 3′end of the first transposon to the 5′ ends of the target fragments toproduce a plurality of 5′ tagged target fragments; treating the solidsupport to remove unbound nucleic acids; or treating the solid supportto remove the transposase from the complex, optionally by (a) heatingthe solid support and/or (b) washing the solid support with an enzymedenaturing agent, wherein the enzyme denaturing agent optionallycomprises sodium dodecyl sulfate (SDS), guanidine hydrochloride, urea,or proteinase; treating the plurality of 5′ tagged target fragments witha polymerase and a ligase to extend and ligate the 5′ tagged targetfragments to produce fully double-stranded tagged fragments, optionallywherein the treating with a polymerase and a ligase is done in thepresence of a DNA secondary structure disruptor, wherein the disruptoris optionally DMSO; removing the fully double-stranded tagged fragmentsfrom the solid support, optionally wherein the removing comprisesapplying heat and/or a denaturant sufficient to cleave the fullydouble-stranded tagged fragments from the solid support, optionallywherein the denaturant is NaOH; and selecting the fully double-strandedtagged fragments using capture beads, optionally wherein the capturebeads are magnetic beads, further optionally wherein two separateselecting steps are performed.

In some embodiments, the immobilized transposome complex comprises asolid support; and a transposome complex immobilized to the solidsupport, wherein the transposome complex comprises a transposase; afirst transposon comprising a 3′ transposon end sequence and an anchorsequence (Anchor); a second transposon comprising a 5′ transposon endsequence and a B15′ sequence; and an attachment polynucleotidecomprising an anchor sequence complement (Anchor′), an A14′ sequence, aspacer, and a P5′ sequence and a binding element comprising biotin,wherein the biotin is immobilized to the solid support. In someembodiments, a method further comprises sequencing one or more of thefully double-stranded tagged fragments.

Methods of Generating Tagged Nucleic Acid Fragments Through CombinedTagmentation and Indexing

In some embodiments, dual-indexed paired-end libraries may be preparedfrom a DNA sample using a combined tagmentation and indexing step. Byavoiding separate tagmentation and indexing steps, this protocol has theadvantage of ease-of-use and shorter duration. Further, this protocolcan avoid a denaturation step and produce double-stranded librarieswithout the need for a separate step to produce double-stranded samplesfrom denatured single-stranded samples. This protocol can also avoidcertain washing steps, further reducing the time required for theworkflow.

This method uses a first immobilized transposon complex and a secondimmobilized transposon complex. In the first immobilized transposoncomplex, sequence X in the first transposon is an anchor sequence, andsequence X′ in the attachment polynucleotide is an anchor sequencecomplement. In the second immobilized transposon complex, sequence X inthe first transposon is an anchor sequence, and sequence X′ in theattachment polynucleotide is an anchor sequence complement. The anchorsequences of the first and second immobilized transposon complexes maybe non-complementary to avoid cross-hybridization. The first transposoncomplex may comprise an exemplary first attachment polynucleotidecomprising (i) an anchor sequence complement, an A14′ sequence, aspacer, and a P5′ sequence, and (ii) a binding element comprisingbiotin, and the second transposon complex may comprise an exemplarysecond attachment polynucleotide comprising (i) an anchor sequencecomplement, a B15′ sequence, a spacer, and a P7′ sequence, and (ii) abinding element comprising biotin.

In combined tagmentation and indexing a suspension of solidsupport-linked transposomes (BLTs, i.e., the immobilized transposoncomplexes) comprising the first and second transposome complexes may beadded to each well. An indexing step may then be performed in a singlereaction solution with Index 1 (i7) adapters, Index 2 (i5) adapters, andsequences required for sequencing cluster generation ligated in the samereaction solution as the tagmentation. The first indexingoligonucleotides may comprise a A14 sequence, i5 sequence, and P5sequence, and second indexing oligonucleotides may comprise a B15sequence, i7 sequence, and P7 sequence. Conditions for thetagmentation/ligation step include tagmentation buffer and E. Coli DNAligase added to the mixture of target DNA, first and second transposomecomplexes, and the first and second indexing oligonucleotides. Thiscombined tagementation and indexing step may proceed for various times,such as from at least 1 minute to at least 15 minutes, or from at least5 minutes to at least 15 minutes.

The tagmentation and indexing reactions may then be stopped using avariety of methods, including heating the solid support and/or washingthe solid support with an enzyme denaturing agent, such as optionallyusing sodium dodecyl sulfate (SDS), guanidine hydrochloride, urea, orproteinase. The time for stopping the reaction, whether by heatingand/or through a washing step, may vary from at least 1 minute to atleast 5 minutes.

The 5′ tagged target fragments ligated to indexing oligonucleotides maybe treated with a polymerase to extend and produce fully double-strandedtagged fragments. The extension reaction may proceed for various times,such as from at least 1 minute to at least 10 minutes, or from at least2 minutes to at least 10 minutes.

Further, library preparation comprising combined tagmentation andindexing may also avoid a qPCR step, as the final product is adouble-stranded DNA library in solution.

In some embodiments, the contacting a first immobilized transposomecomplex and a second immobilized transposome complex and the treatingthe plurality of 5′ tagged target fragments with a ligase are performedin a single reaction.

In some embodiments, the double-stranded tagged fragments are producedin solution.

Some embodiments further comprise selecting the fully double-strandedtagged fragments using capture beads, optionally wherein the capturebeads are magnetic beads, further optionally wherein two separateselecting steps are performed.

In some embodiments, the method further comprises sequencing one or moreof the fully double-stranded tagged fragments.

Target Nucleic Acid

The target nucleic acid can be any type that comprises DNA, RNA, cDNA,or the like. For example, the target nucleic acid may be in a variety ofstates of purification, including purified nucleic acid. In someembodiments, the biological sample comprises a mixture of nucleic acids(such as DNA), protein, other nucleic acid species, other cellularcomponents, and/or any other contaminant, present in approximately thesame proportion as found in vivo. For example, in some embodiments, thecomponents are found in the same proportion as found in an intact cell.Because the methods provided herein allow nucleic acid or DNA to bebound to a solid support through the tagmentation process, othercontaminants can be removed by washing the solid support aftertagmentation occurs. The biological sample can comprise, for example, acrude cell lysate or whole cells. For example, a crude cell lysate thatis applied to a solid support in a method set forth herein, need nothave been subjected to one or more of the separation steps that aretraditionally used to isolate nucleic acids from other cellularcomponents.

Thus, in some embodiments, the biological sample can comprise not onlypurified nucleic acids from any source but also, for example, unpurifiednucleic acids as found in blood, plasma, serum, lymph, mucus, sputum,urine, semen, cerebrospinal fluid, bronchial aspirate, feces, andmacerated tissue, or a lysate thereof, or any other biological specimencomprising nucleic acid or DNA material. Target nucleic acid may be froma tissue sample, tumor sample, cancer cells, or a biopsy sample. Thetarget nucleic acid may be cell-free DNA (cfDNA).

Target nucleic acid may come from any species, of from a mixture ofspecies. For example, target nucleic acid may be from a mammal (such asa human, dog, cat, cow, pig, sheep, or other domesticated animal), orother species such as fish, bacteria, virus, fungus, or archaea. Nucleicacid may come from environmental samples, such as soil or water.

In some embodiments, the target nucleic acid is DNA. In one suchembodiment, the DNA is double-stranded. In some further embodiments, thedouble-stranded DNA comprises genomic DNA. In some other embodiments,the target nucleic acid is RNA or a derivative thereof, or cDNA. In someembodiments, the target nucleic acid is a product of an upstreamreaction, such as an amplification or replication event, for example, anamplicon. In some embodiments, the target nucleic acid is bisulfitetreated DNA.

In some embodiments, a biological sample (raw sample or extract) isprocessed to purify target nucleic acids prior to the tagmentationmethods described herein. In some embodiments, the biological sample isa raw sample or a raw sample lysate (e.g., blood, saliva, cell orcells). In some embodiments, the treatment method comprises providing araw sample, raw sample lysate, or pre-processed sample (e.g., a blood orsaliva sample), mixing the sample with a lysis buffer and proteinase K,incubating the mixture to lyse cells in the sample and release DNA fromthe cells, thereby provided target nucleic acid(s) for the tagmentationmethods described herein. An amount of biological sample is notspecifically required, so long as the biological sample containssufficient nucleic acids for analysis. Thus, an amount of biologicalsample may include from about 1 μL to about 500 μL, such as 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,150, 200, 250, 300, 350, 400, 450, or 500 μL, or an amount within arange defined by any two of the aforementioned values.

Components in raw samples or raw sample lysates such as blood, oradditives in pre-processed samples such as saliva that has beencollected in an Oragene collection tube (stabilization agents incollection tubes), may inhibit tagmentation reactions. Thus, providedherein is a method for treating a raw sample, raw sample lysate, orpre-processed sample to overcome this problem. In some embodiments, themethod comprises providing a raw sample, raw sample lysate, orpre-processed sample (e.g., a blood or saliva sample), mixing the samplewith a lysis buffer, proteinase K, and DNA capture or purification beads(e.g., SPRI beads, beads comprising carboxyl groups, where the beads areoptionally magnetic beads), incubating the mixture to lyse cells in thesample and release DNA from the cells, thereby capturing the DNA on theDNA purification or SPRI beads, and separating the beads comprising thecaptured DNA from the mixture. The separating serves to remove potentialtagmentation inhibitors present in the supernatant. The method furthercomprises optionally washing the beads comprising the captured DNA, andeluting the DNA from the beads to provide target nucleic acid(s).

In some embodiments, the target nucleic acid is present in an amountsufficient for generating a library for sequencing. In some embodiments,a quantity of target nucleic acid is an amount of gDNA of 10-500 ng,such as 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500ng, or an amount within a range defined by any two of the aforementionedvalues. In some embodiments, a target nucleic acid is a gDNA present inan amount of 50 ng.

Methods of Sequencing

Some of the methods provided herein include methods of analyzing nucleicacids. Such methods include preparing a library of template nucleicacids of a target nucleic acid, obtaining sequence data from the libraryof template nucleic acids, and optionally assembling a sequencerepresentation of the target nucleic acid. The DNA fragments produced bytransposome mediated tagmentation can be sequenced according to anysuitable sequencing methodology, such as direct sequencing or nextgeneration sequencing, including sequencing by synthesis, sequencing byligation, sequencing by hybridization, sequencing based on detection ofreleased protons can use an electrical detector and associatedtechniques that are commercially available from Ion Torrent (Guilford,Conn., a Life Technologies subsidiary), nanopore sequencing and thelike. In some embodiments, the DNA fragments are sequenced on a solidsupport, such as a flow cell. Exemplary SBS procedures, fluidic systems,and detection platforms that can be readily adapted for use with nucleicacid libraries produced by the methods of the present disclosure aredescribed, for example, in Bentley et al., Nature 456:53-59 (2008), WO04/018497; U.S. Pat. No. 7,057,026; WO 91/06678; WO 07/123744; U.S. Pat.Nos. 7,329,492; 7,211,414; 7,315,019; 7,405,281, and US 2008/0108082,each of which is incorporated herein by reference.

The methods described herein are not limited to any particular type ofsequencing instrumentation used.

EXAMPLES

The following examples serve to describe but not limit the disclosureprovided herein.

Example 1: No Attachment Oligonucleotide with User Cleavable Linker

An experiment was done to compare PCR-free sequencing data to PCRsequencing data. Libraries were prepared using TruSeq PCR-free, TruSeqNano (+PCR), Nextera Flex (+PCR), and the present method and thensequenced to 25× coverage. There were two replicates with each method,with eight libraries in total. FIG. 7A shows that PCR-free methods(striped bars and checked bars, which represent the present method andTruSeq PCR-free, respectively) have improved indel precision and recallcompared to the methods with PCR (black bars and white bars, whichrepresent TruSeq Nano and Nextera Flex, respectively). FIG. 7B showsthat coverage of GC-rich promoter regions is also improved in thePCR-free methods (bottom two panels, with the present method in thebottom left panel and TruSeq PCR-free in the bottom right panel).

Example 2: Generating a Library of Tagged Nucleic Acid Fragments withoutUsing PCR Amplification

Step A1. Formation of Immobilized Transposome Complexes—Indexed Beads.

A mixture of: (1) first transposon having a 3′ mosaic end (ME) sequence(SEQ ID NO: 6), a first tag sequence (A14), and a 5′ primer sequence(P5′) (25 μM final concentration); (2) a second transposon with a 5′complementary mosaic end sequence (ME′; SEQ ID NO: 3) and a second tagsequence (B15′) (37.5 μM final concentration); and (3) an attachmentpolynucleotide that included a 3′ sequence complementary to the secondtag sequence (B15), an index sequence (in this case, i701 in Table 1),and a 5′ primer sequence (P7) with a biotin at the 5′ terminus (25 μMfinal concentration) were annealed by treating with 10 mM Tris-HCl (pH8.0), 1 mM EDTA, and 25 mM NaCl, heating at 95° C. for 10 min, and thencooling to 10° C. over approximately 15 min. The annealedtransposons/attachment polynucleotide (2 μM final concentration based onattachment polynucleotide) were then mixed with a transposase enzyme(6.1 μM final concentration) and incubated at 37° C. overnight to formsolution-phase transposome complexes (FIG. 4A, indexed bead figures).The solution-phase transposome complexes were then immobilized on solidsupport by mixing with streptavidin-coated beads. In some examples, theimmobilization may be carried out in the presence of HT1 buffer(Illumina), which is a high salt buffer that aids formation ofbiotin-streptavidin bonds. After rotating in HT1 for 1 h, the beads werepelleted and washed once in a mixture of HT1 buffer and 50% glycerolstandard storage buffer (Illumina) (9:1). The beads were thenresuspended in 360 μL of buffer containing 50 mM Tris pH 7.5, 30 mMNaCl, and 0.1% Tween 20. 40 μL of EPX2 (Illumina) was added and thebeads were rotated for a further 10 min at room temperature. The beadswere pelleted again and then resuspended in 396 μL of the same buffer,treated with 4 μL of single-stranded binding protein (5 mg/mL), androtated again for 10 min. The beads were pelleted again, washed once inthe 9:1 HT1:standard storage buffer mixture, and resuspended in 15%glycerol standard storage buffer (Illumina). The resulting indexed beadswere used in single indexing and dual indexing tagmentation protocols.

Step A2. Formation of Immobilized Transposome Complexes—Universal Beads.

Annealed transposons were prepared as described in Step A1, with theexception that the attachment polynucleotide included a 5′ sequencecomplementary to the first tag sequence (A14′) and a 3′ primer sequence(P5′) with a biotin at the 3′ terminus (for single indexing), andoptionally an intervening universal nitroindole sequence (for dualindexing). The transposome complexes were prepared from the annealedtransposons and immobilized on solid support as described for Step A1.The resulting universal beads were used in single indexing and dualindexing tagmentation protocols as indicated.

Step B. Tagmentation.

DNA (e.g., about 50 pg to 5 μg) was mixed with immobilized transposomecomplexes and 50 μL of tagmentation buffer (10 mM Tris acetate (pH 7.6),5 mM magnesium acetate, and 10% dimethylformamide, as described in U.S.Pat. Nos. 9,080,211, 9,085,801, and 9,115,396, each of which isincorporated by reference, and the resulting mixture was incubated at55° C. for 5 min. A library of tagged DNA fragments immobilized on thebeads was generated. The tagmentation reaction mixture was treated with10 of a stop buffer comprising 5% SDS, 100 mM Tris-HCl (pH7.5), 100 mMNaCl, and 0.1% Tween 20, and the resulting mixture was incubated at 37°C. for 5 min to denature the transposase enzyme from the transposomecomplexes. The beads with the immobilized DNA fragments were thenpelleted on a magnet and washed three times in wash buffer (100 mMTris-HCl (pH 7.5), 100 mM NaCl, and 0.1% Tween) to remove any residualSDS. The beads were separated from the supernatant by magnetic captureand were washed further using 100 mM Tris-HCl (pH 7.5), 100 mM NaCl, and0.1% Tween 20. The resulting tagmented fragments for all four approachesare depicted in FIG. 4B.

Step C. Extension, Ligation, and Indexing.

Extension and ligation was performed by contacting the beads with anextension and ligation mix (ELM) that included T4 DNA polymerase(exonuclease minus) and E. coli DNA ligase and incubating at 30° C. for15 min followed by 16° C. for 15 min, thereby gap filling DNA fragmentsbetween the 3′ ends of the fragments and the 5′ ends of the ME′sequences and extending single-stranded regions to generate fullydouble-stranded products by incorporating the remaining sequences fromthe attachment polynucleotide (FIG. 4C). In certain cases, indices werealso added during this step. As shown in FIG. 4D, top right figure, anindex was added to the universal bead/single index construct by addingan oligonucleotide with a double-stranded primer sequence (P7/P7′), adouble-stranded index sequence (in this case, i701/i701′), and asingle-stranded overhang region with a 3′ sequence complementary to thesecond tag sequence (B15). For the indexed beads/dual indexing approach(FIG. 4D, bottom left figure), the index reagent included adouble-stranded primer sequence (P5/P5′), a double-stranded indexsequence (in this case, i501/i501′), and a single stranded overhangregion with a 5′ sequence complementary to the first sequence tag(A14′). For the universal beads/dual indexing approach (FIG. 4D, bottomright figure), the index reagent was the same as for the universalbead/single index case, but an additional second index reagent includinga complementary primer sequence (P5) and an index sequence (i5) washybridized and ligated to the 5′ end of the A14 sequences.

The reaction mixture was treated with 10 μL of a stop buffer comprising5% SDS, 100 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 0.1% Tween 20, andthe resulting mixture was incubated at 37° C. for 5 min to denature thetransposase enzyme from the transposome complexes. The beads with theimmobilized DNA fragments were then pelleted on a magnet and washedthree times in wash buffer (100 mM Tris-HCl (pH 7.5), 100 mM NaCl, and0.1% Tween) to remove any residual SDS. The beads were separated fromthe supernatant by magnetic capture and were washed further using 100 mMTris-HCl (pH 7.5), 100 mM NaCl, and 0.1% Tween 20.

Step D. Release of Tagged Fragments.

After the third wash, the beads were resuspended in 100 μL of 0.2 N NaOHto denature the library fragments and release them from the beads. Thelibrary was purified by adding 100 μL of supernatant directly to 180 μLof SPRI beads, following the standard SPRI purification protocol, andeluting in 15 μL of Illumina resuspension buffer. The libraries werequantified by qPCR, and then prepared for sequencing on a MiSeq® bydiluting to 5 μL of about 3200 pM and denaturing with 5 μL of 0.2 N NaOHat room temperature for 5 minutes. Chilled Illumina HT1 buffer (990 μL)was added and the full 1 mL mixture was loaded into the MiSeq®cartridge.

Results are shown in FIG. 4E, and summarized in Table 2 below, forindexed beads or universal beads with single or dual indexing.

TABLE 2 Results of Library Preparation for Various Transposome ComplexConfigurations Median Reads Bases Sample Insert Clusters Aligned AlignedIndexed Beads, Single 390, 404, 93% 87%, 87%, 85%, 85%, Indexing (3samples) 393 90% 88% Universal Beads, Single 417 84% 53% 83% Indexing (1sample) Indexed Beads, Dual 353, 364, 90% 91%, 90%, 90%, 90%, Indexing(3 samples) 366 88% 90% Universal Beads, Dual 385 91% 68% 86% Indexing(1 sample)

Using the methods described herein, the biases in sequencing data thatare typically introduced with PCR were overcome. For example, using thePCR-free methods described herein, the resulting libraries of nucleicacid fragments were sequenced, and the sequences did not havesignificant gaps in the GC rich regions, as is typically observed whenPCR is used.

Example 3: Sequencing Comparison of Test Libraries

In this example, two comparison libraries from a sample containing aknown 100% GC repeat expansion (FMR1) were prepared using Nextera™ DNAFlex (which includes PCR) and TruSeq™ PCR-Free Library Prep Kits) andfour test libraries were prepared as described herein using theuniversal beads/dual indexing method, as shown in FIG. 4D. As shown inFIG. 8 , no repeats were called in the sequencing data from the libraryprepared using Nextera™ DNA Flex (first column, no bar shown as resultwas 0). Repeats were called using TruSeq™ PCR-Free (column 2) and thefour test libraries using the methods described herein (Samples 1-4,columns 3-6). Thus, the present methods demonstrate improved calling ofrepeat expansion samples with 100% GC regions.

Example 4: Forked Oligonucleotide with Cleavable Linker

Step 1. Formation of Transposome Complexes.

A biotinylated oligonucleotide (50 μM) comprising a 5′ biotin, three Tresidues, then three U residues, P5, A14, and ME, with the sequence:

(SEQ ID NO: 20) /5Biosg/TTUUUAATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAand an oligonucleotide comprising ME′ B15′ P7′ (75 μM) with thesequence:

(SEQ ID NO: 21) CTGTCTCTTATACACATCTCCGAGCCCACGAGACATCTCGTATGCCGTCTTCTGCTTGwas treated with 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 25 mM NaCl,heated to 95° C. for 10 min, and then cooled to 10° C. at −0.1° C. persecond. The annealed transposons were then mixed with a transposaseenzyme so that the final concentration of biotinylated oligo was 2 μMthe transposase concentration was 4 μM. The mixture was incubated at 37°C. overnight to form solution-phase transposome complexes. Thesolution-phase transposome complexes were then immobilized on solidsupport by mixing with streptavidin-coated beads and HT1 buffer(Illumina) and rotating at room temperature for 1 h. The beads were thenwashed three times in HT1 buffer before being resuspended in 15%glycerol standard storage buffer (Illumina) (FIG. 2A).

Step 2. Tagmentation.

DNA (e.g., about 50 pg to 5 μg) was mixed with immobilized transposomecomplexes and 50 μL of tagmentation buffer (10 mM Tris acetate (pH 7.6),5 mM magnesium acetate, and 10% dimethylformamide, as described in U.S.Pat. Nos. 9,080,211, 9,085,801, and 9,115,396, each of which isincorporated by reference, and the resulting mixture was incubated at55° C. for 5 min. A library of tagged DNA fragments immobilized on thebeads was generated. The tagmentation reaction mixture was treated with10 μL of a stop buffer comprising 5% SDS, 100 mM Tris-HCl (pH7.5), 100mM NaCl, and 0.1% Tween 20, and the resulting mixture was incubated at37° C. for 5 min to denature the transposase enzyme from the transposomecomplexes. The beads with the immobilized DNA fragments were thenpelleted on a magnet and washed three times in wash buffer (100 mMTris-HCl (pH 7.5), 100 mM NaCl, and 0.1% Tween) to remove any residualSDS. The beads were separated from the supernatant by magnetic captureand were washed further using 100 mM Tris-HCl (pH 7.5), 100 mM NaCl, and0.1% Tween 20.

Step 3. Extension and Ligation.

Extension and ligation were performed by contacting the beads with anextension and ligation mix (ELM) that included T4 DNA polymerase(exonuclease minus) and E. coli DNA ligase and incubating at 30° C. for15 min followed by 16° C. for 15 min, thereby gap filling DNA fragmentsbetween the 3′ ends of the fragments and the 5′ ends of the ME′sequences. The reaction mixture was treated with 10 μL of a wash buffercomprising 5% SDS, 100 mM Tris-HCl (pH7.5), 100 mM NaCl, and 0.1% Tween20, and the resulting mixture was incubated at 37° C. for 5 min todenature the transposase enzyme from the transposome complexes. Thebeads with the immobilized DNA fragments were then pelleted on a magnetand washed three times in wash buffer (100 mM Tris-HCl (pH 7.5), 100 mMNaCl, and 0.1% Tween) to remove any residual SDS. The beads wereseparated from the supernatant by magnetic capture and were washedfurther using 100 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 0.1% Tween 20.

Step 4. Release of Tagged Fragments.

After the third wash, the beads were resuspended in a buffer containing50 mM potassium acetate, 20 mM tris-acetate, 10 mM magnesium acetate,and 100 μg/mL BSA. 5 μL of an enzyme mixture containing uracil DNAglycosylase and endonuclease VIII were added and the libraries wereincubated at 37° C. for 30 min. The enzymes act together to cleave theU's in the attachment oligonucleotides and release the library fragmentsinto solution. The library was purified by adding 100 μL of supernatantdirectly to SPRI beads, following the standard size selection SPRIpurification protocol (0.5× right and 0.7-0.8× left), and eluting in 15μL of Illumina resuspension buffer. The resulting libraries werequantified by qPCR (FIG. 2B).

Example 5: Modified Extension-Ligation Protocol

An alternative protocol for the indexed variation extension-ligationreaction (Example 2C) may be used. In some instances, extension of thei7 index is inefficient due to secondary structure in certain indexsequences. Thus, for the construct from FIG. 6C, eight different indexpairs were investigated. As shown in FIG. 9A, the % CV of the index poolwas 76%. It was discovered that adding a P7′ oligonucleotide during theextension-indexing reaction boosts the performance of certain indexpairs, perhaps due to hybridization to the P7 sequence that may disruptor prevent secondary structure formation. In this instance, theextension-indexing reaction also includes ligation. P7′ (with 5′phosphate for ligation) is added during the extension-ligation-indexingreaction at 2.5 μM. For the construct shown in FIG. 6D, FIG. 9B showsthe % CV for eight index pairs at less than 30%.

The addition of DMSO to the P7′ oligo extension-ligation-indexingreaction improves % CV further. In this example, for the construct ofFIG. 6D, DMSO is added to the extension-ligation-indexing reaction at5%. The extension-ligation reaction contains all of the necessaryenzymes and components for extension and ligation. The reaction isincubated at 37° C. for 30 min before proceeding with the workflow asnormal. As shown in FIG. 9C, % CV for eight different index pairs wasless than 20%. This result allows the system to be used withoutquantifying DNA output from the library for each sample to adjust forvariations in indexing performance.

Example 6: PCR Free Immobilized Transposome in Tube

This example demonstrates a method and system for immobilizingtransposome complexes in a tube, such as in a PCR tube, for PCR freewhole genome sequencing. The methods are performed similarly to themethod as described in Example 2, and the entire preparation may beperformed in the same tube.

Formation of Immobilized Transposome Complexes in a Tube.

A mixture of: (1) first transposon having a 3′ mosaic end (ME) sequence(SEQ ID NO: 6), a first tag sequence (A14), and a 5′ primer sequence(P5) (25 μM final concentration); (2) a second transposon with a 5′complementary mosaic end sequence (ME′; SEQ ID NO: 3) and a second tagsequence (B15′) (37.5 μM final concentration); and (3) an attachmentpolynucleotide that included a 3′ sequence complementary to the secondtag sequence (B15), an index sequence, and a 5′ primer sequence (P7)with a biotin at the 5′ terminus (25 μM final concentration) wereannealed by treating with 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 25 mMNaCl, heating at 95° C. for 10 min, and then cooling to 10° C. overapproximately 15 min. The attachment polynucleotide included a 5′sequence complementary to the first tag sequence (A14′) and a 3′ primersequence (P5′) with a biotin at the 3′ terminus (for single indexing).In some embodiments, the attachment polynucleotide included anintervening universal nitroindole sequence (for dual indexing). Theannealed transposons/attachment polynucleotides (2 μM finalconcentration based on attachment polynucleotide) were then mixed with atransposase enzyme (6.1 μM final concentration) and incubated at 37° C.overnight to form solution-phase transposome complexes. Thesolution-phase transposome complexes were then immobilized on a surfaceof a PCR tube by mixing the complexes (having biotin) in a PCR tube witha streptavidin-coated surface. In some examples, the immobilization maybe carried out in the presence of HT1 buffer (Illumina, San Diego,Calif.), which is a high salt buffer that aids formation ofbiotin-streptavidin bonds. After incubation for 1 h, the PCR tube waswashed in a mixture of TWB buffer (Illumina, San Diego, Calif.). Theresulting tubes having transposome immobilized thereon were used insingle indexing and dual indexing tagmentation protocols.

The library preparation workflow, including the steps of tagmentation,extension, ligation, and indexing, were performed as outlined in Example2. After completion of the steps as outlined in Example 2, libraryfragments immobilized to the surface of the tube were generated. Thetubes were rinsed with 50 μL of 0.2 N NaOH to denature the libraryfragments and to release them from the surface of the tubes. Thelibraries were quantified by qPCR, and then prepared for sequencing on aMiSeq® by diluting to 5 μL of about 3200 pM and denaturing with 5 μL of0.2 N NaOH at room temperature for 5 minutes. Chilled Illumina HT1buffer (990 μL) was added and the full 1 mL mixture was loaded into theMiSeq® cartridge.

Results of sequencing is shown in FIG. 10 , which shows the coverage ina region of the genome known to be difficult to sequence (gene RNPEPL1).The two library prep methods with PCR (Nextera Flex and tube-basedNextera) show a gap where no sequences covered a region of the gene,indicated with rectangular bounding box. The two PCR-free methods werefound to have good coverage in this region as shown by the sectionsindicated in FIG. 10 as “PCR-free Flex” and PCR-free tube-basedNextera.”

Example 7: PCR-Free Preparation without Cleavable Linker

The following example demonstrates a method and system for immobilizingtransposome complexes in a tube, such as in a PCR tube, for PCR-freewhole genome sequencing. The methods are performed similarly to themethod described in Example 2, but without an attachment nucleotidecomprising a cleavable linker or a step of cleaving the linker. Thismethod employs three separate oligonucleotides. Additional components ofthe method include the Tn5 transposon, SDS, an extension-ligation mix(ELM), and a streptavidin-coated surface (such as beads or a plate).

A mixture of (1) a first transposon having a ME sequence, a first tagsequence (A14), an i5 sequence, and a 5′ primer sequence (P5); (2) asecond transposon with a 5′ complementary mosaic end sequence (ME′), asecond tag (B15′), an i7′ sequence, and a 3′ primer sequence (P7′); and(3) an attachment polynucleotide comprising a P7 sequence and a bindingelement (such as biotin) can be prepared and attached to a surface (suchas a streptavidin-coated plate or bead). The transposome can be builtand attached to the surface in a single step in the presence of templatenucleic acid in tagmentation buffer.

Tagmentation is performed for 5 min at 55° C. PEG may be included in thetagmentation reaction. The mixture is treated with SDS to remove the Tn5transposase. ELM is performed and the supernatant is removed. No SPRIcapture beads are needed with this method, and thus no need to tuneinsert size via bead-loading density. Fragments can be eluted in NaOHfor sequencing preparation.

Example 8: PCR-Free Preparation of 96-Well Libraries

The following example demonstrates a method and system for preparing upto 96 dual-indexed paired-end libraries from a DNA sample. The methodsare performed similarly to the method as described in Example 2, but usetwo steps of incubation with capture beads to allow preparation withouta final qPCR step. As qPCR can be time-consuming (up to two hours), thisprotocol has the advantage of ease-of-use and shorter duration. Thismethod was compatible with DNA inputs of 25-1000 ng. For human DNAsamples and other large complex genomes, DNA input can be greater than200 ng. For DNA input of 200 to 1000 ng, quantification andnormalization of the initial DNA sample is not required. For 25 to 200ng DNA input, libraries are quantified and normalized before sequencing.

This method uses a first transposon as described in FIG. 6D, whereinsequence X in the first transposon is ATCTGACTATCCCCTGCG (SEQ ID NO:23), and sequence X′ in the attachment polynucleotide isCGCAGGGGATAGTCAGAT (SEQ ID NO: 24). The anchor sequence is A14, and thespacer region is made up of 2 C18 and 1 C9 carbon spacers (non-DNAsequences).

Tagmentation.

DNA in 10 mM Tris-HCl (about 2-30 μL) is added to each well of a 96-wellPCR plate so that the total input amount (ng) is within the desiredrange. If the DNA volume is <30 μL, nuclease-free water is added to theDNA samples to bring the total volume to 30 μL. A suspension ofbead-linked transposomes (BLTs) (10 μL) is added to each well followedby 10 μL of tagmentation buffer. The BLTs are loaded on the beads at aconcentration of from about 50 to 1000 nM. They are resuspended in 15%glycerol standard storage buffer, which consists of 15% glycerol, 100 mMNaCl, 50 mM Tris HCl pH 7.5, 0.1 mM EDTA, 1 mM DTT, 0.1% triton X-100.Samples are pipetted to mix until beads are fully resuspended, and theplate is sealed and placed on a thermal cycler at 41° C. for 5 minfollowed by a hold at 10° C. Tagmentation times of 15, 30, 45, 60, 75,90, 105, 120, and 300 sec provide similar yields, with a shift to longerinsert lengths for the shorter incubation times (e.g., 15 sec producedlonger insert sizes than 60, 120, or 300 sec). In some examples,tagmentation time is reduced to 1 min.

To each well is added 10 μL of stop tagmentation buffer comprising SDSand the resulting mixture is pipetted until the beads are fullyresuspended, and the mixture is incubated for 1-5 min. The plate isplaced on a magnetic stand for 2-5 min, and the supernatant is removedand discarded. The plate is removed from the magnet, and 150 μL oftagmentation wash buffer is added directly onto the beads in each well.Samples are mixed by pipette and the plate is placed on a magnetic standuntil the solution is clear (approx. 2-5 min). This step removed SDSfrom the samples.

Extension/Ligation.

Index 1 (i7) adapters, Index 2 (i5) adapters, and sequences required forsequencing cluster generation are added by extension/ligation. While theplate is still on the magnetic stand, the supernatant is removed anddiscarded. The plate is removed from the magnetic stand and 45 μL ofextension ligation mix is added to each well followed by pipetting toresuspend the beads. Next, the appropriate index adapters (5 μL) areadded to each sample, and the plate is sealed and placed on a thermalcycler at 37° C. for 5 min, 50° C. for 5 min, and then at hold at 10° C.The 37° C. and 50° C. incubation periods can each be performed at 1, 3,or 5 min, and all variations produce similar library yields. Shorterincubation periods produce libraries with increased fragment size, whilelonger incubations lead to higher concentrations of ligation products.The plate is then placed on the magnetic stand for 2-5 min and thesupernatant is then removed.

To the wells is added 75 μL of tagmentation wash buffer and the mixtureis pipetted to resuspend the tagmentation beads. The supernatant isdiscarded, and the plate is spun down and placed on the magnet for 2-5min. To each well is added 45 μL of 0.2N NaOH to denature the fragments.The mixture in each well is pipetted to resuspend the tagmentation beadsand the plate is incubated for 1-5 min at room temperature.

Library Clean-Up.

To each well is added 36 μL of capture beads (such as Ampure XP beads)and the well contents were mixed to resuspend the beads and thenincubated for 1, 3, or 5 min. The plate is placed on a magnetic standfor approximately 1-5 min until the supernatant is clear.

To each well of a second 96-well plate is added 42 μL of capture beads.Then, 76 μL of the supernatant from each well of the first 96-well plateis added to the second 96-well plate with capture beads. Samples aremixed by pipette and incubated for 1, 3, or 5 min and then placed on amagnetic stand until supernatant is clear (approximately 1-5 min). Insome examples, the second capture bead step is omitted.

Single or double capture bead clean-up protocols, and 1, 3, or 5 minincubations for each bead clean-up step, are suitable.

Following capture bead purification, the supernatant is discarded andthe capture beads are washed twice with 180 μL of fresh 80% ethanolwithout mixing and incubated at room temperature, after which thesupernatant is discarded. Residual ethanol is removed by pipette, andthe plate is air-dried and removed from the magnetic stand.

To each well is added 15 μL-22 μL of resuspension buffer and wellcontents are mixed by pipetted to resuspend the beads. The plate isincubated at room temperature for 2 min and then placed on a magneticstand until the supernatant is clear (approximately 2 min). A total of14 μL-20 μL of supernatant from each well is transferred to a third96-well PCR plate. This plate is the final DNA library and is preparedfor sequencing as described above.

This protocol employs two purification steps with capture beads, andeliminates a qPCR step, allowing for completion of the librarypreparation in a shorter time. For example, samples are prepared usingthese methods in under 30 min when using short magnet incubation stepstotaling 20 min.

The protocol can be performed in other vessels, such as microcentrifugetubes.

In some embodiments, to achieve optimal cluster density, equal libraryvolumes are pooled and the pool quantified before sequencing. When using200-1000 ng DNA inputs, in some instances, libraries are combined bypooling equivolumes from each library in a 1.7 mL microcentrifuge tube,vortexing to mix, and then centrifuging. The library pool is quantifiedusing an ssDNA Qubit kit to determine the concentration of the pool.When using 25-200 ng DNA inputs, in some examples, each library isquantified individually using an ssDNA Quibit kit.

In some examples, sequencing is done on a NovaSeq6000 cartridge.

Example 9: Preparation of Libraries Via Combined Tagmentation andIndexing

The following example demonstrates a method and system for preparingdual-indexed paired-end libraries from a DNA sample using a combinedtagmentation and indexing step. The methods were performed similarly tothe method as described in Example 2, but combined tagmentation andindexing in one step. By avoiding separate tagmentation and indexingsteps, this protocol has the advantage of ease-of-use and shorterduration. Further, this protocol can avoid a denaturation step andproduce double-stranded libraries without the need for a separate stepto produce double-stranded samples from denatured single-strandedsamples. This protocol can also avoid certain washing steps, furtherreducing the time required for the workflow.

To show that these libraries can be prepared using reactions that aregiven either a long, medium, or short period of time to proceed, threesets of samples can be processed at a normal workflow rate, a fastworkflow rate, and a superfast workflow rate, respectively.

This method uses a first immobilized transposon complex and a secondimmobilized transposon complex. In the first immobilized transposoncomplex, sequence X in the first transposon is an anchor sequence, andsequence X′ in the attachment polynucleotide is an anchor sequencecomplement. In the second immobilized transposon complex, sequence X inthe first transposon is an anchor sequence, and sequence X′ in theattachment polynucleotide is an anchor sequence complement. The anchorsequence comprised in the first and second immobilized transposoncomplexes may be non-complementary to avoid cross-hybridization. Thefirst transposon complex may comprise an exemplary first attachmentpolynucleotide comprising (i) an anchor sequence complement, an A14′sequence, a spacer, and a P5′ sequence, and (ii) a binding elementcomprising biotin, and the second transposon complex may comprise anexemplary second attachment polynucleotide comprising (i) an anchorsequence complement, a B15′ sequence, a spacer, and a P7′ sequence, and(ii) a binding element comprising biotin.

Tagmentation and Indexing. DNA in 10 mM Tris-HCl (about 2-30 μL) isadded to each well of a 96-well PCR plate so that the total input amount(ng) is within the desired range. A suspension of bead-linkedtransposomes (BLTs, i.e., the immobilized transposon complexes)comprising the first and second transposome complexes is added to eachwell. An indexing step is performed in a single reaction solution withIndex 1 (i7) adapters, Index 2 (i5) adapters, and sequences required forsequencing cluster generation ligated in the same reaction solution asthe tagmentation. In this example, first indexing oligonucleotidescomprises a A14 sequence, i5 sequence, and P5 sequence, and secondindexing oligonucleotides comprises a B15 sequence, i7 sequence, and P7sequence. Conditions for the tagmentation/ligation step includetagmentation buffer and E. Coli DNA ligase added to the mixture oftarget DNA, first and second transposome complexes, and the first andsecond indexing oligonucleotides for a total volume of 20 μL. Thetagmentation and indexing reaction is conducted at a temperature of 41°C. for three different time intervals (for three different samples): 15minutes (normal workflow), 5 minutes (fast workflow), and 1 minute(superfast workflow).

Following the combined tagmentation and indexing reaction, to each wellis added 5 μL of 0.6% SDS with an incubation at 37° C. to stop thetagmentation reaction by washing with SDS to denature the transposase.The stopping step was conducted for three different time intervals (forthree different samples): 5 minutes (normal workflow), 5 minutes (fastworkflow), and 1 minute (superfast workflow). While the stopping step isconducted for the same period of time for the normal and fast workflow,the overall time for the fast workflow is still shorter due to timedifferences in the other steps.

Extension. To each well is added 75 μL of extension mix (DNA polymerase,dNTPs, and buffer), and the extension reaction proceeds at <68° C. in a100 μL reaction volume. The extension step is conducted for threedifferent time intervals (for three different samples): 10 minutes(normal workflow), 2 minutes (fast workflow), and 1 minute (superfastworkflow). This extension step can generate a double-stranded DNAlibrary in solution. Library clean-up may be performed with capturebeads.

Overall, library yields of the normal and fast workflow are comparableto methods that had separate tagmentation and indexing steps, while thelibrary yields of the superfast workflow are likely sufficient for manyuses, especially considering the added convenience of the even fasterworkflow. Methods with combined tagmentation and indexing may allowindexing oligonucleotides to associate with transposases that did notsuccessfully fragment the DNA sample (i.e., side tagmentation products).Further, combined tagmentation and indexing may generate libraryproducts having P5/P5 sequences or P7/P7 sequences at both ends, andthese library products with homozygous ends may not sequence properly.However, for many applications, the amount of starting DNA sample issufficient to enable sequencing results even if some side products aregenerated.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made. Accordingly, otherembodiments are within the scope of the following claims.

SEQUENCE LISTING SEQ ID NO: 1 - A14-ME TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGSEQ ID NO: 2 - B15-ME GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGSEQ ID NO: 3 - ME′ phos-CTGTCTCTTATACACATCT SEQ ID NO: 4 - Al4TCGTCGGCAGCGTC SEQ ID NO: 5 - B15 GTCTCGTGGGCTCGG SEQ ID NO: 6 - MEAGATGTGTATAAGAGACAG SEQ ID NO: 7 - P5 AATGATACGGCGACCACCGAGAUCTACACSEQ ID NO: 8 - P7 CAAGCAGAAGACGGCATACGAG*A SEQ ID NO: 9 - P5_A14_MEAATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGT GTATAAGAGACAGSEQ ID NO: 10 - P_ME′_B15′ 5Phos/CTGTCTCTTATACACATCTCCGAGCCCACGAGACSEQ ID NO: 11 - Bio_P7_i701_B1_5_ddC5Biosg/CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGG CTCGG/3ddC/SEQ ID NO: 12 - A14′_P5′_bioGACGCTGCCGACGAGTGTAGATCTCGGTGGTCGCCGTATCATT/3Bio/SEQ ID NO: 13 - P_A14_ME 5Phos/TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGSEQ ID NO: 14 - A14′_nitro_P5′_bioGACGCTGCCGACGACCC/i5NitInd//i5NitInd//i5NitInd//i5NitInd//i5NitInd//i5NitInd//i5NitInd//i5NitInd/GTGTAGATCTCGGTGGTCGCCGTATCATT/3Bio/SEQ ID NO: 15 - A14′_12anc′_2sp_P5′_bioGACGCTGCCGACGACCGAGCATATCC/iSp18//iSp18/GTGTAGATCTCGGTGGTCGCCGTATCATT/3BioTEG/ SEQ ID NO: 16 - P5_i501AATGATACGGCGACCACCGAGATCTACACTAGATCGC SEQ ID NO: 17 - P_i701′_P7_ddC5Phos/TAAGGCGAATCTCGTATGCCGTCTTCTGCTTG/3ddC/SEQ ID NO: 18 - P7_i701_B15_ddCCAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGG/ 3ddC/SEQ ID NO: 19 - P5_i501_12ancAATGATACGGCGACCACCGAGATCTACACTAGATCGCGGATATGCTCGGSEQ ID NO: 20 - biotinylated oligonucleotide/5Biosg/TTUUUAATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTGTATAAGAGACA SEQ ID NO: 21 - ME′_B15′_P7′CTGTCTCTTATACACATCTCCGAGCCCACGAGACATCTCGTATGCCGTC TTCTGCTTGSEQ ID NO: 22 - Exemplary anchor sequence GGATATGCTCGGSEQ ID NO: 23: Exemplary sequence X ATCTGACTATCCCCTGCGSEQ ID NO: 24: Exemplary sequence X′ CGCAGGGGATAGTCAGAT

What is claimed is:
 1. An annealed transposon/attachment polynucleotidehybrid comprising: (a) a first transposon comprising a 3′ transposon endsequence and a 5′ adaptor sequence; (b) a second transposon comprising a5′ transposon end sequence and a 3′ adaptor sequence, wherein i. thesecond transposon 5′ transposon end sequence is complementary to thefirst transposon 3′ transposon end sequence, forming a double-strandedtransposon end sequence to which the transposase is bound, ii. the 5′adaptor sequence of the first transposon and the 3′ adaptor sequence ofthe second transposon are non-complementary, and iii. the firsttransposon and the second transposon form a Y-shape; and (c) anattachment polynucleotide comprising: i. an attachment adaptor sequencehybridized to either the first transposon 5′ adaptor sequence or thesecond transposon 3′ adaptor sequence; and ii. a biotin linker.
 2. Atransposome complex comprising: (a) a transposase; (b) a firsttransposon comprising a 3′ transposon end sequence and a 5′ adaptorsequence; (c) a second transposon comprising a 5′ transposon endsequence and a 3′ adaptor sequence, wherein i. the second transposon 5′transposon end sequence is complementary to the first transposon 3′transposon end sequence, forming a double-stranded transposon endsequence to which the transposase is bound, ii. the 5′ adaptor sequenceof the first transposon and the 3′ adaptor sequence of the secondtransposon are non-complementary, and iii. the first transposon and thesecond transposon form a Y-shape; and (d) an attachment polynucleotidecomprising: i. an attachment adaptor sequence hybridized to either thefirst transposon 5′ adaptor sequence or the second transposon 3′ adaptorsequence and ii. a biotin linker.
 3. An immobilized transposome complexcomprising: a solid support; and a transposome complex immobilized tothe solid support, wherein the transposome complex comprises: (a) atransposase; (b) a first transposon comprising a 3′ transposon endsequence and a 5′ adaptor sequence; (c) a second transposon comprising a5′ transposon end sequence and a 3′ adaptor sequence, wherein i. thesecond transposon 5′ transposon end sequence is complementary to atleast a portion of the first transposon 3′ transposon end sequence,forming a double-stranded transposon end sequence to which thetransposase is bound, ii. the 5′ adaptor sequence of the firsttransposon and the 3′ adaptor sequence of the second transposon arenon-complementary, and iii. the first transposon and the secondtransposon form a Y-shape; and (d) an attachment polynucleotidecomprising: i. an attachment adaptor sequence hybridized to either thefirst transposon 5′ adaptor sequence or the second transposon 3′ adaptorsequence and ii. a biotin linker, wherein the biotin linker isimmobilized to the solid support.
 4. The immobilized transposome complexof claim 3, wherein the biotin linker comprises a cleavable linker. 5.The transposome complex of claim 2, wherein the attachmentpolynucleotide further comprises a spacer region, an anchor region, aprimer sequence, or an index tag sequence, or a combination thereof, andoptionally further comprises a capture sequence, a barcode sequence, acleavage sequence, or a sequencing-related sequence, or a combinationthereof, optionally wherein: (a) the first transposon further comprisesa primer sequence 5′ of the 5′ adaptor sequence, and the attachmentpolynucleotide comprises (i) a portion complementary and hybridized tothe 5′ adaptor sequence and (ii) a sequence that is complementary to theprimer sequence; (b) the first transposon further comprises a primersequence 5′ of the 5′ adaptor sequence, and the attachmentpolynucleotide comprises a sequence that is complementary to the primersequence; (c) the second transposon comprises the 3′ adaptor sequenceand the attachment polynucleotide comprises (i) a portion complementaryand hybridized to the 3′ adaptor sequence, (ii) an index tag sequence,and (iii) a primer sequence; and/or (d) the attachment polynucleotidecomprises a spacer region or a spacer region and an anchor region,optionally wherein the first transposon comprises the 5′ adaptorsequence, and the attachment polynucleotide comprises (i) a portioncomplementary and hybridized to the 5′ adaptor sequence, (ii) a spacerregion, and (iii) a primer sequence.
 6. The transposome complex of claim2, wherein the attachment polynucleotide comprises an A14′ sequence. 7.The transposome complex of claim 5, wherein the spacer region a) is anon-DNA spacer; b) is a length of approximately 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 15, 20, or 30 base pairs, optionally wherein the spacer regionis a length of approximately 8 or 10 base pairs or nucleotides; c)comprises universal bases, optionally wherein the universal basescomprise an inosine or a nitroindole sequence; and/or d) comprises oneor more C18 spacers, one or more C9 spacers, or a combination thereof,optionally wherein the spacer region comprises a 2×sp18 syntheticlinker, two C18 spacers, a C9 spacer, or two C18 spacers and one C9spacer.
 8. The transposome complex of claim 5, wherein the anchor regionis complementary to an anchor region complement of an indexingoligonucleotide, where the indexing oligonucleotide comprises the anchorregion complement and an index tag sequence (-Anchor′-Index TagSequence-), optionally wherein the anchor region complement is common toa plurality of indexing oligonucleotides and/or each index tag sequenceof a plurality of indexing oligonucleotides is the same or different. 9.The transposome complex of claim 2, wherein the transposase is a Tn5transposase, a wild-type Tn5 transposase, or a hyperactive Tn5transposase, or a mutant thereof; and/or the primer sequence of theattachment polynucleotide is complementary to an index primer sequenceof the indexing polynucleotide.
 10. The immobilized transposome complexof claim 3, wherein the solid support is a bead, a paramagnetic bead, aflow cell, a surface of a microfluidic device, a tube, a well of aplate, a slide, a patterned surface, or a microparticle, optionallywherein the solid support comprises a plurality of solid supports,optionally wherein the plurality of solid supports comprises a pluralityof beads.
 11. A kit comprising: a transposome complex of claim 2; and anindex mix comprising index sequences, optionally wherein the index mixcomprises i5 index sequences and i7 index sequences, wherein the i5index sequences hybridize to the attachment polynucleotide, and whereinthe i7 index sequences hybridize to the 3′ adaptor sequence.